Isoxazole-4-carboxamide compounds active against protein tryosine kinase related disorders

ABSTRACT

The present invention relates to novel isoxazole-4-carboxamides which modulate the activity of protein tyrosine kinases and therefore are expected to be useful in the treatment of abnormal protein tyrosine kinase activity driven disorders including cancer.

This application claims benefit of U.S. Provisional Application No.06/047,084, filed May 19, 1997.

INTRODUCTION

The present invention relates generally to organic chemistry,biochemistry, pharmacology and medicine. More particularly, it relatesto novel heterocyclic compounds, and their physiologically acceptablesalts, which modulate the activity of protein tyrosine kinases which areinvolved in the control of cell proliferation, differention and growthand therefore are expected to exhibit a salutary effect againstdisorders related to abnormal protein tyrosine kinase activity.

BACKGROUND OF THE INVENTION

The following is offered as background information only and is notadmitted to be prior art to the present invention.

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. Growth factor receptors (“Gfrs”) are an importantpart of the signal transduction pathway. Gfrs are cell-surface proteins.When bound by a growth factor ligand, Gfrs are converted to an activeform which interacts with proteins on the inner surface of a cellmembrane. As the result of this interaction, one of the key biochemicalmechanisms of signal transduction is initiated; i.e., the reversiblephosphorylation of various proteins within the cell. Thisphosphorylation of intra-cellular proteins causes the formation insidethe cell of complexes with a variety of cytoplasmic signaling moleculesthat, in turn, effect numerous cellular responses such as cell division(proliferation), cell differentiation, cell growth, expression ofmetabolic effects to the extracellular microenvironment, etc. For a morecomplete discussion, see Schlessinger and Ullrich, Neuron, 9:303-391(1992). See also, Posada and Cooper, Mol. Biol. Cell., 3:583-392 (1992)and Hardie, Symp. Soc. Exp. Biol., 44:241-255 (1990).

The molecules which effect the phosphorylation of proteins are calledprotein kinases (“PKs”). One of the classes of PKs, which is ofparticular importance to the present invention, phosphorylates proteinson the alcohol moiety of serine, threonine and tyrosine residues ineukariotic cells. These PKs fall essentially into two groups, thosespecific for phosphorylating serines and threonines, and those specificfor phosphorylating tyrosines. The protein tyrosine kinases (“PTKs”) canbe further divided into receptor PTKs, abbreviated “receptor tyrosinekinases” or “RTKs” and non-receptor PTKs, sometimes refered to as“cellular tyrosine kinases” or “CTKs.”

The RTKs are comprised of an extracellular glycosylated ligand bindingdomain, a transmembrane domain and an intracellular cytoplasmiccatalytic domain that can phosphorylate tyrosine residues on proteins.On the other hand CTKs are entirely intra-cellular and do not containextracellular and transmembrane domains.

PTKs play an important role in the control of cellular processesincluding proliferation, differentiation, migration and survival.Enhanced PTK activity due to activating mutations or overexpression hasbeen implicated in many human cancers. It is clear from numerous studies(q.v, infra) that the activity of PTKs must be tightly controlled innormal cells and healthy tissue, as mutations resulting in overactivityof PTKs cause diseases that are associated with excessive cell growthand proliferation while mutations which result in reduction or loss ofactivity can cause, e.g., embryonal lethality or developmentaldisorders.

The RTKs comprise one of the larger families of PTKs and have diversebiological activity. At present, at least nineteen (19) distinctsubfamilies of RTKs have been identified. One such subfamily is the“HER” family of RTKs, which include EGFR (epithelial growth factorreceptor), HER2, HER3 and HER4. These RTKs consist of an extracellularglycosylated ligand binding domain, a transmembrane domain and anintracellular cytoplasmic catalytic domain that can phosphorylatetyrosine residues on proteins. One well-known example of the apparentinvolvement of PTKs/RTKs in cellular disorders is the association ofHer2 over-expression with breast cancer (Slamon, et al., Science,244:707 (1989).

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and the insulin receptor relatedreceptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II toform a heterotetramer of two entirely extracellular glycosylated αsubunits and two β subunits which cross the cell membrane and whichcontain the tyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fms. These receptors consist of glycosylated extracellulardomains composed of variable numbers of immunoglobin-like loops and anintracellular domain wherein the tyrosine kinase domains is interruptedby unrelated amino acid sequences.

Another group which, because of its similarity to the PDGFR subfamily,is sometimes subsumed in the later group is the fetus liver kinase(“flk”) receptor subfamily. This group is believed to be made of up ofkinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R,flk-4 and fms-like tyrosine kinase 1 (flt-1).

Finally, the FGFR family of PTKs contains at least four distinctmembers: FGFR1 (also called Flg and Cek1), FGFR2 (also called Bek, Ksam,KsamI and Cek3), FGFR3 (also called Cek2) and FGFR4. They share a commonstructure consisting of, in the mature protein, one or moreimmunoglobulin-like (IgG-like) loops flanked by characteristiccysteines, a hydrophobic transmembrane domain and a intracellular domaincontaining a catalytic region that is split by a short insert; SeeUllrich and Schlessinger, Cell, 61:203 (1990).

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl,Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

Both RTKs and CTKs have been implicated in a host of pathogenicconditions including, significantly, cancer.

Other pathogenic conditions to which RTKs and CTKs have been linkedinclude, without limitation, psoriasis, hepatic cirrhosis, diabetes,atherosclerosis, arterial restinosis, kidney sclerosis, wound scarringand a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PTK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs) and cytoplasmic PTKs (CTKs), discussed above.

In view of the apparent link between PTK-related cellular activities anda number of virulent human disorders, it is no surprise that a greatdeal of effort is being expended in an attempt to identify ways tomodulate PTK activity. Some of these have involved biomimetic approachesusing large molecules patterned on those involved in the actual cellularprocesses (e.g., mutant ligands (U.S. App. No. 4,966,849)); solublereceptors and antibodies (App. No. WO 94/10202, Kendall and Thomas,Proc. Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature,362:841-844 (1993)); RNA ligands (Jelinek, et al., Biochemistry,33:10450-56); Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, etal., Exp. Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys.,152:448-57)) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660;WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al.,Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).

More recently, attempts have been made to identify small molecules whichact as PTK inhibitors. For example, bis-monocylic, bicyclic andheterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindolederivatives (PCT WO 94/14808 and 1-cyclopropyl-4-pyridylquinolones (U.S.Pat. No. 5,330,992) have been described as tyrosine kinase inhibitors.Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP App.No. 0 566 266 A1), selenaindoles and selenides (PCT WO 94/03427),tricyclic polyhydroxylic compounds (PCT WO 92/21660) andbenzylphosphonic acid compounds (PCT WO 91/15495) have all beendescribed as compounds for use as PTK inhibitors for use in treatment ofcancer.

SUMMARY OF THE INVENTION

Our own efforts to identify small organic molecules which modulate PTKactivity and which, therefore, should be useful in the treatment andprevention of disorders driven by abnormal PTK activity, has led us tothe discovery of novel heteroarylcarboxamide compounds which modulatePTK activity and which are the subject of this invention.

Thus, in one aspect, the present invention relates to novelheteroarylcarboxamides which modulate the activity of PTKs. In addition,the present invention relates to the preparation and use ofpharmacological compositions of the disclosed compounds and theirphysiologically acceptable salts in the treatment and prevention of PTKdriven disorders.

As used herein, a “heteroarylcarboxamide” refers generally to a“C-amido” group, as defined herein, where the “C” carbon is covalentlybonded to a carbon atom of a “heteroaryl” group, also as defined herein,R¹² is hydrogen and R¹³ is selected from the “aryl” and “heteroaryl”groups defined, infra.

It is understood that when Z, q.v., infra, is sulfur, the compound willformally be a heteroarylthiocarboxamide; however, whenever the termheteroarylcarboxamide is used herein, it will refer to the sulfur analogas well.

A “pharmacological composition” refers to a mixture of one or more ofthe compounds described herein, or a physiologically acceptable saltthereof, with other chemical components, such as pharmaceuticallyacceptable carriers and excipients. The purpose of a pharmacologicalcomposition is to facilitate administration of a compound to anorganism.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

An “excipient” refers to an inert substance added to a pharmacologicalcomposition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

1. THE COMPOUNDS

4-(N-(4′-trifluoromethylphenyl)carboxamido)-5-methyl-isoxazole(“leflunomide”, Formula 1), to which the compounds of the presentinvention are structurally related, is a compound currently in clinicaltesting, based on its ability to inhibit unwanted cell proliferation,both as an immunosuppressive and a cancer drug. Leflunomide is believedto be metabolized in serum such that the isoxazole ring is converted toan open form called in the literature A771726 and having the chemicalstructure shown in Formula 2.

Several early reports suggested that leflunomide was capable ofinhibiting tyrosine kinase signaling (Bartlett, et al., Agents andActions, 32:10 (1991), Mattar, et al., FEBS Lett., 334:161 (1993), Xu,et al., J. Biol. Chem., 270:12398 (1995)). More recently Cherwinski, etal. reported that leflunomide has no effect on tyrosine kinase signalingbut inhibited proliferation by inhibiting DNA replication and entry ofcells into M-phase of the cell cycle (J. Pharma. and Exp. Thera.,272:460 (1995)). Subsequent reports have suggested that the activity ofleflunomide is solely due to the ability of A771726 to inhibitpyrimidine biosynthesis by inhibiting a key enzyme in that process,dihydro-orotate dehydrogenase (DHOD) (Nair, et al., Imm. Lett., 47:171(1995), Greene, et al., Biochem. Pharma., 50:861 (1995), Cherwinski, etal., Inflamm. Res., 44:317 (1995), Davis et al., Biochem., 35:1270(1996)). Thus it has been widely accepted in the art that leflunomideacts only as a prodrug.

The ability of A771726 to inhibit pyrimidine biosynthesis is overcome bythe addition of uridine which is characteristic of pyrimide biosynthesisinhibitors. The compounds of the present invention, on the other hand,while structurally similar to lefunomide, are capable of inhibitingcellular growth by a mechanism not affected by the addition of uridine.While not being bound to any particular theory, applicants believe thatis is due either to fact that the heteroaryl group of the claimedcompounds do not metabolize to an open form at all and therefore exhibittheir activity in their native configuation, or, if they do metabolizeto an open form, the chemical composition of the open form molecules towhich they are converted are either inactive or active but not asinhibitors of pyrimidine biosynthesis (as evinced by the fact thaturidine addition has no effect).

Thus, it appears that, while chemically similar to leflunomide, thecompounds of the present invention are biologically active in anentirely different manner than leflunomide and its metabolite, A771726,and therefore comprise a new family of compounds capable of modulatingprotein tyrosine kinase activity.

While, again, not being bound to a particular theory, it appears thatthe compounds of this invention affect cell proliferation by modulatingPTK signaling. The signaling related to the PTKs FGFR and PDGFR appearto be particularly susceptible to modulation by the compounds of thepresent invention.

As used herein, the terms “modulate”, “modulation” or “modulating” referto the alteration of the catalytic activity of PTKs. In particular,modulating refers to the activation of the catalytic activity of PTKs,more preferably the activation or inhibition of the catalytic activityof PTKs, depending on the concentration of the compound administered or,more preferably still, the inhibition of the catalytic activity of PTKs.Modulation may be effected by direct interaction with a PTK or throughintervention at some other point in the biochemical process controlledby the particular PTK, the observable result of which appears as amodulation of PTK catalytic activity.

The term “catalytic activity” as used herein refers to the rate ofphosphorylation of tyrosine under the influence, direct or indirect ofPTKs.

A. General Structural Features.

In a second aspect, the present invention relates toheteroarylcarboxamide compounds having the chemical structure shown inFormula 3:

A is selected from the group consisting of oxygen, nitrogen and sulfur.

B is selected from the group consisting of nitrogen and sulfur and it isunderstood that when B is sulfur and A is nitrogen, the nitrogen isparticipating in both a single bond and a double bond within the ring sothat it cannot be bonded to any atom outside the ring; that is, when Bis sulfur, R² cannot exist.

D, E, F, G, and J are independently selected from the group consistingof carbon and nitrogen such that the monocyclic heteroaryl six-memberring formed is one known in the chemical arts; furthermore, when D, E,F, G or J is nitrogen, R⁵, R⁶, R⁷, R⁸ or R⁹, respectively, does notexist.

R¹ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl and heteroalicyclic.

R² is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, carbonyl,C-carboxy, S-sulfonamido, sulfonyl, hydroxy, alkoxy,trihalomethanesulfonyl, halo, guanyl, C-amido and C-thioamido.

R³ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl and heteroalicyclic.

Z is selected from the group consisting of oxygen and sulfur.

R⁴ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl heteroaryl, heteroalicyclic, sulfonyl,trihalomethanesulfonyl, hydroxy, alkoxy and C-carboxy.

R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, alkenyl, alkynyl,cycycloalkyl, aryl, heteroaryl, heteroalicyclic hydroxy, alkoxy,cycloalkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy,thioalkyoxy, thiocycloalkoxy, thioheteraryloxy, thioheteralicycloxy,halo, nitro, cyano, C—O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, silyl, phosphonyl, C-carboxy, O-carboxy, N-amido,C-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,trihalomethanesulfonyl, guanyl, guanidino, trihalomethanesulfonamido,amino and —NR¹³R¹⁴.

R¹³ and R¹⁴ are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, carbonyl, C-carboxy, sulfonyl,trihalomethanesulfonyl and, combined, a five- or six-memberheteroalicyclic ring containing at least one nitrogen.

As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms (whenever anumerical range; e.g. “1-20”, is stated herein, it means that the group,in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms,3 carbon atoms, etc. up to and including 20 carbon atoms). Morepreferably, it is a medium size alkyl having 1 to 10 carbon atoms. Mostpreferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkylgroup may be substituted or unsubstituted. When substituted, thesubstituent group(s) is preferably one or more individually selectedfrom trihaloalkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic,hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy,thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano,halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl,sulfonyl, sulfonamido, trihalomethanesulfonamido,trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido, phosphonyl,amino and NR¹³R¹⁴.

R¹³ and R¹⁴ are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, carbonyl, C-carboxy, sulfonyl,trihalomethysulfonyl and, combined, a five- or six-memberheteroalicyclic ring containing at least one nitrogen.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more individually selectedfrom alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,heteroaryloxy, heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy,thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl, sulfonyl,sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl,guanyl, guanidino, ureido, phosphonyl, amino and NR¹³R¹⁴ with R¹³ andR¹⁴ as defined above.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more selectedfrom alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,alkoxy, aryloxy, heteroaryloxy, heteroalicylcoxy, thiohydroxy,thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicyloxy, cyano,halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl,sulfonyl, sulfonamido, trihalomethanesulfonamido,trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido, phosphonyl,amino and NR¹³R¹⁴ with R¹³ and R¹⁴ as defined above.

As used herein, a “heteroaryl” group refers to a monocyclic or fusedring (i.e., rings which share an adjacent pair of atoms) group having inthe ring(s) one or more atoms selected from the group consisting ofnitrogen, oxygen and sulfur and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofheteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline,purine and carbazole. The heteroaryl group may be substituted orunsubstituted. When substituted, the substituted group(s) is preferablyone or more selected from alkyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy,heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy,thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl, sulfonyl,sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl,guanyl, guanidino, ureido, phosphonyl, amino and NR¹³R¹⁴ with R¹³ andR¹⁴ as defined above.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms selected from the groupconsisting of nitrogen, oxygen and sulfur. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. The heteroalicyclic ring may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably one or more selected from alkyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy,heteroalicylcoxy, thiohydroxy, thioalkoxy, thioaryloxy,thioheteroaryloxy, thioheteroalicyloxy, cyano, halo, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfinyl, sulfonyl,sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl,guanyl, guanidino, ureido, phosphonyl, amino and NR¹³R¹⁴ with R¹³ andR¹⁴ as defined above.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “heteroaryloxy” group refers to a heteroaryl-O— group with heteroarylas defined herein.

A “heteroalicycloxy” group refers to a heteroalicyclic-O— group withheteroalicyclic as defined herein.

A “thiohydroxy” group refers to an —SH group.

A “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “thioheteroaryloxy” group refers to a heteroaryl-S— group withheteroaryl as defined herein.

A “thioheteroalicycloxy” group refers to a heteroalicyclic-S— group withheteroalicyclic as defined herein.

A “carbonyl” group refers to a —C(═O)—R″ group, where R″ is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon), as each is definedherein.

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as definedherein.

A “trihalomethanecarbonyl” group refers to a X₃CC(═O)— group with X asdefined herein.

A “C-carboxy” group refers to a —C(═O)O—R″ groups, with R″ as definedherein.

An “O-carboxy” group refers to a R″C(═O)O— group, with R″ as definedherein.

A “carboxylic acid” group refers to a C-carboxyl group in which R″ ishydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein.

A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above.

A “trihalomethanesulfonamido” group refers to a X₃CS(═O)₂NR¹³— groupwith X and R¹³ as defined herein.

A “cyano” group refers to a —C═N group.

A “sulfinyl” group refers to a —S(═O)—R″ group, with R″ as definedherein.

A “sulfonyl” group refers to a —S(═O)₂R″ group, with R″ as definedherein.

An “S-sulfonamido” group refers to a —S(═O)₂NR¹³R¹⁴, with R¹³ and R¹⁴ asdefined herein.

An “N-Sulfonamido” group refers to a R¹³S(═O)₂NR¹⁴— group, with R¹³ andR¹⁴ as defined herein.

An “O-carbamyl” group refers to a —OC(═O)NR¹³R¹⁴ group with R¹³ and R¹⁴as defined herein.

An “N-carbamyl” group refers to a R¹³OC(═O)NR¹⁴ group, with R¹³ and R¹⁴as defined herein.

An “O-thiocarbamyl” group refers to a —OC(═S)NR¹²R¹³ group with R¹² andR¹³ as defined herein.

An “N-thiocarbamyl” group refers to a R¹²OC(═S)NR¹³— group, with R¹² andR¹³ as defined herein.

An “amino” group refers to an —NH₂ group.

A “C-amido” group refers to a —C(═O)NR¹²R¹³ group with R¹² and R¹³ asdefined herein.

An “N-amido” group refers to a R¹²C(═O)NR¹³— group, with R¹² and R¹³ asdefined herein.

A “C-thioamido” group refers to a —C(═S)NR¹²R¹³ group with R¹² and R¹³and defined herein.

A “ureido” group refers to a —NR¹²C(═O)NR¹³R¹⁴ group, with R¹² and R¹³as defined herein and R¹⁴ defined the same as R¹² and R¹³.

A “guanidino” group refers to a —R¹²NC(═N)NR¹³R¹⁴ group, with R¹², R¹³and R¹⁴ as defined herein.

A “guanyl” group refers to a R¹²R¹³NC(═N)— group, with R¹² and R¹³ asdefined herein.

A “nitro” group refers to a —NO₂ group.

A “cyano” group refers to a —C≡N group.

A “silyl” group refers to a —Si(R″)₃, with R″ as defined herein.

A “methylenedioxy” group refers to a —OCH₂O— group where the two oxygensare covalently bonded to two adjacent carbon atoms of an aryl orheteroaryl ring with aryl and heteroaryl as defined herein.

A “1,3-dioxano” group refers to a —CH₂OCH₂O— group where the —CH₂ andthe oxygen are covalently bonded to two adjacent carbon atoms of an arylor heteroaryl ring with aryl and heteroaryl as defined herein.

Examples of monocyclic heteroaryl six-member rings known in the chemicalarts include, but are not limited to, the following:

B. Preferred Structural Features.

Preferred structural features for the claimed compounds are those inwhich:

A is oxygen and B is nitrogen;

R¹ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl and alkynyl; and,

R³ is selected from the group consisting of hydrogen, alkyl, cycloalkyland aryl; and,

R⁴ is hydrogen.

Further preferred structures for the claimed compounds are those inwhich:

A and B are nitrogen;

R² is selected from the group consisting of hydrogen, alkyl andcycloalkyl;

Z is oxygen;

R⁵, R⁸ and R⁹ are hydrogen;

R⁶ is selected from the group consisting or hydrogen and alkyl; and,

R⁷ is selected from the group consisting of hydrogen, trihalomethyl andtrihalomethanesulfonyl.

Other preferred embodiments of the present invention are those in which:

R⁶ and R⁷, combined, form a methylenedioxy or a 1,3-dioxano group.

And, finally, a preferred structure for the claimed compounds is that inwhich J is nitrogen.

2. THE BIOCHEMISTRY

In yet another embodiment, this invention relates to a method for thetreatment or prevention of a disorder characterized by inappropriate PTKactivity comprising administering to a patient inflicted with such adisorder a therapeutically effective amount of one or more of thedisclosed compounds or a physiologically acceptable salt thereof.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures by,practitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

As used herein, the terms “prevent”, “preventing” and “prevention” referto a method for barring an organism from in the first place acquiring anPTK mediated cellular disorder.

As used herein, the terms “treat”, “treating” and “treatment” refer to amethod of alleviating or abrogating a PTK mediated cellular disorderand/or its attendant symptoms. With regard particularly to cancer, theseterms simply mean that the life expectancy or an individual affectedwith a cancer will be increased or that one or more of the symptoms ofthe disease will be reduced.

As used herein, the term “cancer” refers to various types of malignantneoplasms, most of which can invade surrounding tissues, and maymetastasize to different sites, as defined by Stedman's MedicalDictionary 25th edition (Hensyl ed. 1990). Examples of cancers which maybe treated by the present invention include, but are not limited to,brain, ovarian, colon, prostate, kidney, bladder, breast, lung, oral andskin cancers which exhibit inappropriate PTK activity. These types ofcancers can be further characterized. For example, brain cancers includeglioblastoma multiforme, anaplastic astrocytoma, astrocytoma,ependymoma, oligodendroglioma, medulloblastoma, meningioma, sarcoma,hemangioblastoma, and pineal parenchymal. Skin cancers include melanomaand Kaposi's sarcoma.

A “disorder characterized by inappropriate PTK activity” includes, butis not limited to, cell proliferative disorders, cell differentiationdisorders, cell growth disorders and metastatic disorders. Suchdisorders include, but are not limited to, cancer, as described aboveand, in addition, the development of neoplasia such as carcinoma,sarcoma, leukemia, glioblastoma and hemangioma as well as disorders suchas psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (andother disorders related to uncontrolled angiogenesis and/orvasculogenesis), fibrotic disorders and metabolic disorders.

Unwanted cell proliferation can result from inappropriate PTK activityoccurring in different types of cells including cancer cells, cellssurrounding a cancer cell (stromal cells), endothelial cells and smoothmuscle cells. For example, and without limitation, an increase in FGFRand/or PDGFR activity of endothelial cells surrounding cancer cells maylead to an increased vascularization (angiogenesis) of a tumor, therebyfacilitating growth of the cancer cells. Inappropriate PTK activity mayalso contribute to the proliferation of cancer cells by direct mitogenicstimulation.

“Cell proliferative disorders” refer to disorders wherein unwanted cellproliferation of one or more subset of cells in a multicellular organismoccurs resulting in harm (e.g., discomfort or decreased life expectancy)to the multicellular organism. Cell proliferative disorders can occur indifferent types of animals and in humans. Cell proliferative disordersinclude cancers, skeletal disorders, angiogenic or blood vesselproliferative disorders, fibrotic disorders and mesangial cellproliferative disorders.

Blood vessel proliferative disorders refer to angiogenic andvasculogenic disorders generally resulting in abnormal proliferation ofblood vessels. The formation and spreading of blood vessels, orvasculogenesis and angiogenesis, respectively, play important roles in avariety of physiological processes such as embryonic development, corpusluteum formation, wound healing and organ regeneration. They also play apivotal role in cancer development. Other examples of blood vesselproliferation disorders include arthritis, where new capillary bloodvessels invade the joint and destroy cartilage, and ocular diseases,like diabetic retinopathy, where new capillaries in the retina invadethe vitreous, bleed and cause blindness. Conversely, disorders relatedto the shrinkage, contraction or closing of blood vessels, such asrestenosis, are also implicated.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. Hepatic cirrhosis cancause diseases such as cirrhosis of the liver. An increasedextracellular matrix resulting in a hepatic scar can also be caused byviral infection such as hepatitis. Lipocytes appear to play a major rolein hepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases, such asglomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes, transplant rejection, andglomerulopathies. The PDGF-R has been implicated in the maintenance ofmesangial cell proliferation. Floege et al., 1993, Kidney International43:47S-54S.

Other examples of cell proliferative disorders are disclosed in thefollowing references which are incorporated as if fully set forthherein. EGFRs (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et al.,1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science244:707-712) and the PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633)are over-expressed in many tumors and/or persistently activated byautocrine loops. In fact, in the most common and severe cancers, thesereceptor over-expressions (Akbasak and Suner-Akbasak et al., 1992, J.Neurol. Sci., 111:119-133; Dickson et al., 1992, Cancer Treatment Res.61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070;Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated with breast, ovarian,gastric, lung, pancreas and bladder cancer. PDGFR has been associatedwith glioblastoma, lung, ovarian, melanoma and prostate. The RTK c-methas been generally associated with hepatocarcinogenesis and thushepatocellular carcinoma. Additionally, c-met has been linked tomalignant tumor formation. More specifically, the RTK c-met has beenassociated with, among other cancers, colorectal, thyroid, pancreaticand gastric carcinoma, leukemia and lymphoma. Additionally,over-expression of the c-met gene has been detected in patients withHodgkins disease, Burkitts disease, and the lymphoma cell line.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, integrally involved in the normal growth anddifferentiation of the nervous system, appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of the IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts, the stemcells of the bone marrow) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series ofrecent publications, Baserga even suggests that IGF-IR plays a centralrole in the mechanisms of transformation and, as such, could be apreferred target for therapeutic interventions for a broad spectrum ofhuman malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga,1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol.,14:4588-4595.

The term “organism” refers to any living entity comprised of at leastone cell. A living organism can be as simple as, for example, a singleeukariotic cell or as complex as a mammal, including a human being.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor; (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis; (3) inhibiting to some extent(that is slowing to some extent, preferably stopping) tumor growth;and/or, (4) relieving to some extent (or preferably eliminating) one ormore symptoms associated with the cancer.

A “therapeutically effective amount”, in reference to the treatment of acell proliferative disorder other than a cancer refers to an amountsufficient to bring about one or more of the following results: inhibitthe growth of cells causing the disorder, relieve discomfort due to thedisorder, or prolong the life of a patient suffering from the disorder.

The association between abnormal PTK activity and disease are notrestricted to cancer. For example, RTKs have been associated withmetabolic diseases like psoriasis, diabetes mellitus, wound healing,inflammation, and neurodegenerative diseases. For example, EGFRinvolvment has been indicated in corneal and dermal wound healing.Defects in the Insulin-R and the IGF-1R are indicated in type-IIdiabetes mellitus. A more complete correlation between specific RTKs andtheir therapeutic indications is set forth in Plowman et al., DN&P7:334-339 (1994).

As noted previously, not only RTKs but CTKs as well including, but notlimited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk(reviewed by Bolen et al., FASEB J., 6:3403-3409 (1992)) are involved inthe proliferative and metabolic signal transduction pathway and thuswere expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been demonstrated to be an oncoprotein(pp60^(v-src)) in chicken. Moreover, its cellular homolog, theproto-oncogene pp60^(c-src) transmits the oncogenic signals of manyreceptors. For example, over-expression of EGFR or HER2/neu in tumorsleads to the constitutive activation of pp60^(c-src), which ischaracteristic of the malignant cell but absent in the normal cell. Onthe other hand, mice deficient in the expression of c-src exhibit anosteopetrotic phenotype, indicating a key participation of c-src inosteoclast function and a possible involvement in related disorders.Similarly, Zap70 is implicated in T-cell signaling.

In particular, the compounds of the present invention are expected to beuseful in the treatment and prevention of cell proliferative disorderscharacterized by inappropriate activity of an FGFR, PDGFR or relatedreceptors.

i. FGFR, PDGFR and Related Receptors.

As discussed above, the FGFR family contains at least four distinctmembers: FGFR1 (also called Flg and Cek1), FGFR2 (also called Bek, Ksam,KsamI and Cek3), FGFR3 (also called Cek2) and FGFR4. They share a commonstructure consisting of, in the mature protein, one or moreimmunoglobulin-like (IgG-like) loops flanked by characteristiccysteines, a hydrophobic transmembrane domain and a intracellular domaincontaining a catalytic region that is split by a short insert. (SeeUllrich and Schlessinger, Cell 61:203, 1990.) The degree of homologyvaries between them, with the highest homology being found in thecatalytic domain. Additional diversity in the family is created throughsplice variants that vary the number and character of the IgG-likeregions in the extracellular domain. At least nine FGFR ligands havebeen identified including FGF1 (acidic FGF), FGF2 (basic FGF), FGF3(int-2), FGF4 (Kaposi FGF), FGF5, FGF6, FGF7 (keratinocyte growth factor(KGF)), FGF8 (androgen-induced growth factor) and FGF9. Multiple membersof the FGF ligand family can bind to the same receptor species. For ageneral review of FGFs and FGFRs see Johnson and Williams, Adv. inCancer Res. 60:1, 1993.

The PDGF receptor family, on the other hand, contains only the twoisoforms PDGFR-alpha and PDGFR-beta, which are known to heterodimerize.PDGF ligand is a pleiotropic factor that exists as a homo- orheterodimer of two polypeptides, the A- and B-chains (Habenicht, et al.,Klin. Wochen-Schrift 68:53, 1990; Heldin, EMBO J. 11:4251, 1992). Othertyrosine kinase receptors structurally and functionally related to FGFRand PDGFR include Flt (de Vries, et al., Science 255:989, 1992) and KDR(Terman, et al., BBRC 187;1579, 1992), both of which are activated bythe ligand VEGF (Rosenthal, et al., Growth Factors, 4:53-59, 1990; Conn,et al., Proc. Natl. Acad. Sci. (USA), 87:1323-1327, 1990; Houck, et al.,Mol. Endocrinol., 5:1806-1814, 1991). VEGF expression is known to beincreased by hypoxia (such as would be found in growing tumors) and isknown to stimulate endothelial cells and to be involved in angiogenesis(Plate et al., Nature, 359:845-848, 1992; Shweike, et al., Nature359:843-845, 1992).

PDGFR- and FGF-dependent signaling is initiated immediately followingbinding of ligand to a receptor. Ligand binding induces receptordimerization, either homodimers or heterodimers, leading to activationof receptor tyrosine kinase activity and autophosphorylation. Activationof the receptor leads to increased tyrosine phosphorylation on a numberof cellular proteins, although many of their identities and functionsare still largely unknown. Depending on the cell type, PDGFR and/or FGFRactivation ultimately leads to proliferation, differentiation,inhibition of differentiation, motility, etc.

The use of the present invention is facilitated by first determiningwhether a disorder is related to inappropriate PDGFR and/or FGFRactivity. Once such disorders are identified, patients suffering fromsuch a disorder can be identified by analysis of their symptoms byprocedures well known to medical doctors. Such patients can then betreated as described herein.

Many well known techniques exist for determining whether a disorder isrelated to inappropriate PDGFR and/or FGFR activity. For example,comparisons can be made in the level of expression of FGF and/or PDGFligand or FGFR and/or PDGFR in a tumor biopsy with levels in similarnormal tissues or tumor cells known to be unrelated to FGFR and/or PDGFRactivity (such as A431 cells, Yaish, et al., Science 242:933, 1988).Such comparisons can be done by immunostaining with FGFR and/or PDGFRspecific antibodies or binding and detecting FGF and/or PDGF ligandusing techniques well known in the art, by Northern blot analysis forthe presence of ligand or receptor RNA, or by transcript imaging(Plowman, WO96/34985, published Nov. 7, 1996, and incorporated byreference herein). Alternatively, samples can be analyzed for level ofreceptor phosphorylation, which is indicative of activity, compared tonormal tissues. Receptor phosphorylation is readily detected by meanswell known in the art such as by using anti-phosphotyrosine antibodies.If the cancer cells have a higher level of FGFR and/or PDGFR activity orexpression than non-FGFR and/or PDGFR driven cancers or normal tissues,also preferably a level equal to or greater than previously identifiedFGFR and/or PDGFR driven cancers, then the cancer cells are candidatesfor treatment using the compounds discussed herein.

In the case of cell proliferative disorders arising from to unwantedproliferation of non-cancer cells, the level of receptor activity iscompared to that level occurring in the general population (e.g., theaverage level occurring in the general population of people or animalsexcluding those people or animals suffering from a cell proliferativedisorder). If the unwanted cell proliferation disorder is characterizedby a higher receptor level than that occurring in the generalpopulation, then the disorder is a candidate for treatment using thecompounds described herein.

ii. FGFR- and PDGFR-related Disorders

One class of PTK disorders which involves FGFR and/or PDGFR is the cellproliferative disorders. As discussed above, proliferative disordersresult in unwanted cell proliferation of one or more subset of cells ina multicellular organism resulting in harm to the organism. Two ways inwhich inappropriate PTK/FGFR/PDGFR activity can stimulate unwanted cellproliferation of a particular type of cell are by directly stimulatinggrowth of the particular cell or by increasing vascularization of aparticular area (angiogenesis), such as tumor tissue, therebyfacilitating growth of the tissue. Angiogenesis also plays a significantrole in metastasis, a complex disorder which is discussed in more detailbelow.

Cell proliferative disorders include cancers, blood vessel proliferationdisorders, skeletal malformations and fibrotic disorders. Thesedisorders are not necessarily independent. For example, fibroticdisorders may be related to, or overlap with, blood vessel disorders.For example, Moyamoya disease (which is characterized herein as a bloodvessel disorder) results in the abnormal formation of fibrous tissue inthe intracranial arteries.

Not all cancers found in a particular location within the body will betreatable by the method of the invention, only those characterized byinappropriate FGFR, PDGFR or related receptor activity. For example,only 30% of bladder carcinomas are characterized as highly invasive andprone to metastasis (Raghavan, et al., NEJM 322:1129, 1990; Allen andMaher, J. Cell. Physiol. 155:368, 1993), and these have been associatedwith FGFR activity (Allen and Maher, supra). Breast cancers areassociated with inappropriate FGFR activity in 12%-32% of cases (Adnane,et al., Oncogene 6:659, 1991; Penault-Llorca, et al., Int. J. Cancer61:170, 1995). Friess, et al., (Chirug 65:604, 1994) report thatapproximately 50% of primary pancreatic cancers surveyed express FGFR,and this expression was associated with tumor aggressiveness as measuredby significantly shorter post-operative survival. Holm, et al. (Int. J.Oncology 9:1077, 1996) found expression of PDGFR in only 30% ofnon-small cell lung cancer cell lines examined, and only 10% expressedKDR. Seymour, et al. (supra) found expression of PDGF in 43% of tumorsfrom breast cancer patients. Expression of PDGF correlated with areduced chance of survival. One can determine which cancers aretreatable by the compounds and methods of the invention by employing thetechniques described above for determining inappropriate FGFR and/orPDGFR activity.

The formation and spreading of blood vessels, or vasculogenesis andangiogenesis respectively, play important roles in a variety ofphysiological processes such as embryonic development, wound healing andorgan regeneration. They also play a role in cancer development. Bloodvessel proliferation disorders refer to angiogenic and vasculogenicdisorders generally resulting in abnormal proliferation of bloodvessels. Examples of such disorders, besides cancer, include Moyamoyadisease and macular degeneration. FGFR and KDR have been recognized hashaving a regulatory role in angiogensis, along with other factors, dueto their role in both endothelial cell proliferation and migration(Friesel and Maciag, FASEB J. 9:919, 1995; Folkman and Klagsbrun,Science 235:442, 1987; Mullins and Rifkin, J. Cell. Physiol. 119:247,1984; Gualandris, et al., Cell Growth & Diff. 7:147, 1996). FGFRactivity has been suggested to play a role specifically in theangiogenesis associated with macular degeneration (Amin, et al., Invest.Ophthal. and Vis. Sci. 35(8):3178, 1994). PDGF and VEGF expression isassociated with angiogeneis and metastasis in breast cancer (Anan, etal., Surgery 119:333, 1996). PDGF expression has been correlated withincreased blood vessel count in colon cancers (Hsu, et al., J. Cell.Physiol. 165:239, 1995). PDGF and FGF have been shown to inducesecretion of VEGF by glioma cells (Tsai, et al., J. Neurosurg. 82:864,1995).

Moyamoya disease is characterized by intracranial carotid arterystenosis and occlusions and a fine network of vessels at the base of thebrain; it thus may be described as both an angiogenic and fibroticdisorder (Suzuki and Kodama, Stroke 14:104, 1983; Suzuki and Takaku,Arch. Neurol. 20:288, 1969). Suzui, et al., (Neurosurgery 35(1):20,1994) have found that both FGF ligand and FGFR are increased in thesuperficial temporal artery of patients with Moyamoya disease.

Fibrotic disorders refer to the abnormal formation of extracellularmatrix. Examples of fibrotic disorders include those found in the liver(hepatic cirrhosis), kidney (glomerular sclerosis, interstitialnephritis), lung (interstitial pulmonary fibrosis), arteries(restenosis, atherosclerosis) and skin (wound scarring, scleroderma).

Hepatic cirrhosis is characterized by an increase in extracellularmatrix constituents resulting in the formation of a hepatic scar.Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. Anincreased extracellular matrix resulting in a hepatic scar can also becaused by viral infection such as hepatitis. Lipocytes appear to play amajor role in hepatic cirrhosis. Inappropriate FGFR activity canstimulate lipocyte proliferation.

As noted above, other proliferative disorders involving FGFR, PDGFR andother PTKs can be identified by standard techniques, as well as bydetermination of the efficacy of action of the compound describedherein.

iii. Metastatic Disorders

Liotta, et al. describe invasion and metastasis as “the mostlife-threatening aspects of the oncogenic process” (See review inLiotta, et al., Cell 64:327, 1991). Invasion and metastasis are complexevents mediated by a group of coordinated cellular processes. They arefacilitated by proteins that stimulate tumor cell attachment to anextracellular matrix, proteolysis of barriers such as the basementmembrane, migration into the circulatory system and attachment to andcolony formation in distant organs. There is also a significantcorrelation between metastatic potential and angiogeneic potential, andthe two processes may have many factors in common. (See Claffey, et al.,Cancer Res. 56:172, 1996; Takahashi, et al., Cancer Res. 55:3964, 1995.)

The cellular responses induced by FGF, PDGF and VEGF ligands includethose necessary for metastasis: proliferation, migration, production ofproteases, and neovascularization. FGFR and PDGFR expression have beenassociated with increased aggressiveness and metastasis of a number ofcancers. Nakamoto, et al. (Cancer Research 52:571, 1992) compared FGFand FGFR expression and responsiveness in several human prostate cancercell lines. The degree of metastasis in murine models shown by each ofthe cell lines (LNCaP, DU145 and PC3) correlated directly with FGFRexpression (with LNCaP being the lowest and PC3 being the highest),although the biological consequences of FGFR expression were not studiedin depth. Allen and Maher, J. Cell. Phys. 155:368, 1993) report asimilar study with bladder carcinoma cell lines. In a comparison ofinvasive and non-invasive tumors the invasive tumor (EJ) showed asignificant increase in FGFR at both the protein and RNA level, with thenon-invasive tumor, (RT4) showing almost no FGFR present. The FGFR wasalso shown to be biologically active by receptor phosphorylation inresponse to ligand. Anan, et al. (supra) found PDGF mRNA was expressedmore frequently in breast tumors with lymph node metastases that inthose without metastases.

In addition to all of the above, both RTKs and CTKs are currentlysuspected as being involved in hyperimmune disorders.

This invention is therefore directed to compounds which modulate PTKsignal transduction by affecting the enzymatic activity of the PTKs andthereby interfering with the signal transduced by such proteins. Moreparticularly, the present invention is directed to compounds whichmodulate the PTK-mediated signal transduction pathways as a therapeuticapproach to cure many kinds of solid tumors, including but not limitedto carcinoma, sarcoma, leukemia, erythroblastoma, glioblastoma,meningioma, astrocytoma, melanoma and myoblastoma. Indications mayinclude, but are not limited to, brain cancers, bladder cancers, ovariancancers, gastric cancers, pancreas cancers, colon cancers, bloodcancers, lung cancers, bone cancers and leukemias.

The term “administering” as used herein refers to a method forintroducing a compound of this invention into a milieu containing a PTK,including both in vitro, i.e. in a test tube, and in vivo, i.e. intocells or tissues of a living organism, milieus. Thus, the PTK mediateddisorders which are the object of this invention can be studied,prevented or treated by the methods set forth herein whether the cellsor tissues of the organism exist within the organism or outside theorganism. Cells existing outside the organism can be maintained or grownin cell culture dishes. In this context, the ability of a particularcompound to affect a PTK related disorder; i.e., the IC50 of thecompound, defined below, can be determined before the use of thecompounds in more complex living organisms is attempted. For cellsoutside the organism, multiple methods exist, and are well-known tothose skilled in the arts, to administer compounds including, but notlimited to, cell micro-injection, transformation and numerous carriertechniques. For cells harbored within a living organism, myriad methodsalso exist, and are likewise well-known to those skilled in the art, toadminister compounds including, but not limited to, oral, parenteral,dermal, injection and aerosol applications.

As used herein, “PTK related disorder,” “PTK driven disorder”, “abnormalPTK activity” and “inappropriate PTK activity” all refer to a disordercharacterized by inappropriate activity or over-activity of PTKs, whichcan be either RTKs or CTKs. Inappropriate activity refers to either: (1)PTK expression in cells which normally do not express PTKs; (2)increased PTK expression leading to unwanted cell proliferation,differentiation and/or growth; or, (3) decreased PTK expression leadingto unwanted reductions in cell proliferation, differentiation and/orgrowth. Overactivity of PTKs refers to either amplification of the geneencoding a particular PTK or production of a level of PTK activity whichcan correlate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PTK increases, the severity ofone or more of the symptoms of the cellular disorder increases).

The methods and compositions of the invention are designed to inhibitunwanted cell proliferation or metastasis by altering the activity ofPTKs. Without being bound to any theory, inhibition of the activity ofPTKs may occur by inhibiting tyrosine phosphorylation of a RTK, byinhibiting substrate or adaptor protein binding to the receptor, or byinhibiting other downstream signaling events, thereby inhibiting theactivity of the RTK. However, unless otherwise stated, the use of theclaimed methods and compositions are not limited to this particulartheory.

C. Pharmacological Compositions and Therapeutic Applications

The compounds disclosed herein are preferably administered to a patientin a pharmaceutical composition comprising one or more compounds of thisinvention together with pharmaceutically acceptable carrier(s) and/orexcipients. The compounds can be prepared as a physiologicallyacceptable salts (i.e., non-toxic salts which do not prevent thecompound from exerting its effect).

Physiologically acceptable salts can be acid addition salts such ashydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate,tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, cyclohexylsulfamate and quinate. These salts can bederived using acids such as hydrochloric acid, sulfuric acid, phosphoricacid and sulfamic acid, acetic acid, citric acid, lactic acid, tartaricacid, malonic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,and quinic acid.

Physiologically acceptable salts can be prepared by standard techniques.For example, the free base form of the compound is first dissolved in asuitable solvent such as water or a water-alcohol solution containingthe appropriate acid. The salt is then isolated by evaporating thesolution. In a another example, the salt is prepared by reacting thefree base and acid in an organic solvent.

Carriers or excipient can be used to facilitate administration of thecompound, for example, to increase the solubility of the compound.Examples of carriers and excipient include calcium carbonate, calciumphosphate, various sugars or types of starch, cellulose derivatives,gelatin, vegetable oils, polyethylene glycols and physiologicallycompatible solvents. The compositions or pharmaceutical composition canbe administered by different routes including intravenously,intraperitoneally, subcutaneously, intramuscularly, orally, topically,or transmuccosally.

The specific delivery route of any selected agent depends on the use ofthe agent. Generally, a specific delivery program for each agent focuseson agent uptake with regard to intracellular localization, followed bydemonstration of efficacy. Alternatively, delivery to these same cellsin an organ or tissue of an animal can be pursued. Uptake studiesinclude uptake assays to evaluate, e.g., cellular nucleic acid orprotein uptake, regardless of the delivery vehicle or strategy. Suchassays also determine the intracellular localization of the agentfollowing uptake, ultimately establishing the requirements formaintenance of steady-state concentrations within the cellularcompartment containing the target sequence (nucleus and/or cytoplasm).Efficacy and cytotoxicity can then be tested. Toxicity not only includescell viability but also cell function. Generally, the dosages of themutated protein and nucleic acid is as described above for the featuredcompounds.

Drug delivery vehicles are effective for both systemic and topicaladministration. They can be designed to serve as a slow releasereservoir, or to deliver their contents directly to the target cell. Anadvantage of using direct delivery drug vehicles is that multiplemolecules are delivered per uptake. Such vehicles increase thecirculation half-life of drugs which would otherwise be rapidly clearedfrom the blood stream. Some examples of such specialized drug deliveryvehicles falling into this category are liposomes, hydrogels,cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.Pumps can also be used for this purpose.

From this category of delivery systems, liposomes are the preferredapproach. Liposomes increase intracellular stability, increase uptakeefficiency and improve biological activity. Liposomes are hollowspherical vesicles composed of lipids arranged in a similar fashion tothose lipids making up the cell membrane. They have an internal aqueousspace for entrapping water soluble compounds and range in size from 0.05to several microns in diameter. Antibodies can be attached to liposomesto target particular cells.

Topical administration of the featured compound is advantageous,particularly when treating skin disorders such as Kaposi's sarcoma,since it allows localized concentration at the site of administrationwith minimal systemic adsorption. This simplifies the delivery strategyof the agent to the disease site and reduces the extent of toxicologicalcharacterization. Furthermore, the amount of material applied is farless than that required for other administration routes.

Many compounds are preferably absorbed systemically when used to treatdisorders such as cancer. Systemic absorption refers to the accumulationof drugs in the blood stream followed by distribution throughout theentire body. Administration routes which lead to systemic absorptioninclude: intravenous, subcutaneous, intraperitoneal, intranasal andintrathecal. Each of these administration routes expose the drug to anaccessible diseased tissue. Subcutaneous administration drains into alocalized lymph node and proceeds through the lymphatic network into thecirculatory system. The rate of entry into the circulatory system hasbeen shown to be a function of molecular weight or size.

Some of the compounds of this invention may be hydrophobic and thus notvery soluble in water. Effective doses of hydrophobic compounds forsystemic administration can be obtained using the pharmaceuticalformulations described in U.S. Pat. No. 5,610,173, issued Mar. 11, 1997.A particularly preferred formulation is obtained using a combination ofthe compound and VPD:D5W. VPD consists of a solution of 12% w/vpolysorbate 80, 0.55% citric acid (anhydrous), 35% w/v polyenthleneglycol (MW=300 daltons) and 26.3% v/v 190 proof ethanol. VPD is diluted1:22 in a diluent. Preferred diluents are 0.45% saline, and 0.9% saline.A particularly preferred diluent is 5% dextrose in water (D5W).

Another way of overcoming the hydrophobicity problem includes the use offrequent small daily doses rather than a few large daily doses. Forexample, the composition can be administered at short time intervals,preferably the composition can be administered using a pump to controlthe time interval or achieve continuously administration. Suitable pumpsare commercially available (e.g., the ALZET® pump sold by Alzacorporation, and the BARD ambulatory PCA pump sold by Bard MedSystems).

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in an admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used, the dosing regimenand the size and physiological condition of the patient. For thetreatment of cancers it is prefered that the minimal plasmaconcentration in a patient be greater than 5 μg/ml, more preferablygreater than 25 μg/ml, most preferably greater than 50 μg/ml. Thecompound can be delivered daily or less frequently provided plasmalevels of the active moiety are sufficient to maintain therapeuticeffectiveness. Plasma levels may be reduced if pharmacological effectiveconcentrations of the drug are achieved at the site of interest.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals; that is the LD₅₀ (the dose lethal to 50% of the population) andthe ED₅₀ (the dose therapeutically effective in 50% of the population)can be determined. The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal disruption of the proteincomplex, or a half-maximal inhibition of the cellular level and/oractivity of a complex component). Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”,Ch. 1 p1).

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the oncogenic disorder of interest will varywith the severity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

4. SYNTHESIS

The compounds of this invention may be readily synthesized usingtechniques well known in the chemical arts. The following syntheses areshown by way of example only and are not to be construed as limiting inany way. In fact, it will be appreciated by those skilled in the artthat other synthetic pathways for forming the compounds of the inventionare available and that the following are but a few of the possibleroutes to the claimed compounds.

a. 3-Methyl-4-[4-(trifluoromethyl)phenyl-aminocarbonyl]isoxazole.

Ethyl propiolate (2.8 g) and pyrrolidine (1.4 g) in 5 mL of acetonitrileare mixed at room temperature for 1 hour, the solvent evaporated and theethyl 3-pyrrolidin-1-acrylate used as isolated or distilled undervacuum. Triethylamine (0.25 mL) is added to a mixture of 1.8 g of ethyl3-pyrrolidin-1-acrylate, 0.9 g of nitroethane and 2.5 g of phenylisothiocyanate in 10 mL of toluene at room temperature and stirredovernight. The mixture is then refluxed for 0.5 hour, cooled, and thediphenylurea removed by filtration. The mixture is washed with water andbrine, dried over anhydrous sodium sulfate, and evaporated to drynessunder vacuum to give 1.4 g of ethyl 3-methyl-4-isoxazolecarboxylate.(Stork, G., McMurry, J. C., J. Am. Chem. Soc. 89, 5461,1967).

Ethyl 3-methyl-4-isoxazolecarboxylate (1.3 g) is stirred at roomtemperature overnight in 5 mL of ethanol and 10 mL of 2.5 N sodiumhydroxide. Dilution with water, cooling in ice, and acidification to pH2 with 6 N hydrochloric acid precipitates an off-white solid which iscollected by vacuum filtration, washed with ethanol/water, and driedunder vacuum to give 1.0 g of 3-methyl-4-isoxazolecarboxylic acid.

3-Methyl-4-isoxazolecarboxylic acid (0.9 g) is stirred with 5 mL ofthionyl chloride at room temperature for one hour and the mixtureevaporated to dryness. The residue is dissolved in 5 mL tetrahydrofuranand 1 mL of pyridine containing 1.2 g of 4-trifluoromethylaniline andstirred overnight. The mixture is refluxed for one hour, cooled anddiluted with water to give an off-white precipitate. The solid iscollected by vacuum filtation, washed with ethanol/water and dried undervacuum to give 1.2 g of3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole.

b. 3-Methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole.

A solution of 5.5 g of 4-(trifluoromethyl)aniline in 6 mL of toluene at120° C. is treated with 5.4 g of 2,2,6-trimethyl-4H-1,3-dioxin-4-one.The mixture is refluxed for four hours and cooled. The precipitate iscollected by vacuum filtration, washed with toluene and dried to give 6g of N-[4-(trifluoromethyl)phenylacetoacetamide.

N-[4-(Trifluoromethyl)phenylacetoacetamide (6 g), 4 g oftriethylorthoformate and 8 g of acetic anhydride is cautiously heated toreflux for 2 hours. The reaction is cooled to room temperature and theprecipitate collected by vacuum filtration and dried to give 4 g ofN-[4-(trifluoromethyl)phenyl]-2-(ethoxymethylene)acetoacetamide.

N-[4-(Trifluoromethyl)phenyl]-2-(ethoxymethylene)-acetoacetamide (4 g)is suspended in 10 mL of ethanol treated with 1.5 g of hydroxylaminehydrochloride in 10 mL of water which has been adjusted to pH 5 withsodium hydroxide. The mixture is stirred and warmed to 40° C. for 2hours, then cooled to room temperature and the precipitate collected byvacuum filtration. Sodium hydroxide (1 g) is added to the filtrate whichis stirred for 30 minutes. The mixture is acidified to pH 2 with 6 Nhydrochloric acid and the precipitate collected by vacuum filtration.The filtrate is then diluted with 100 mL of water and allowed to standat 4° C. overnight. The precipitate is collected by vacuum filtration,washed with ethanol:water 2:1 and dried to give 300 mg of crude3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole. The crudeis purified on a column of silica gel eluting with ethyl acetate:hexane1:4 to give 100 mg of3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole, anoff-white solid.

c. 3-Methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole.

N-[4-(Trifluoromethyl)phenyl]-2-(ethoxymethylene)acetoacetamide (1 g) in3 mL of ethanol is stirred overnight with 0.3 g of hydrazine hydrate.The precipitate is collected by vacuum filtration, washed withethanol:water 2:1 and dried to give 0.3 g of3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole as anoff-white solid.

d. 3-Methyl-4-(pyrid-2-aminocarbonyl)isoxazole.

By substituting 2-aminopyridine for 4-(trifluoromethyl)aniline in a, theidentical process gives 1 g of3-methyl-4-(pyrid-2-aminocarbonyl)isoxazole as an off-white solid.

e. 3-Methyl-4-(pyrid-2-aminocarbonyl)pyrazole.

By substituting 2-aminopyridine for 4-(trifluoromethyl) aniline in thefirst two steps of the process of b, the identical process gives 2.5 gof N-(pyrid-2-yl)-2-(ethoxymethylene)acetoacetamide. By substitutingN-(pyrid-2-yl)-2-(ethoxymethylene)acetoacetamide forN-[4-(trifluoromethyl)phenyl]-2-ethoxymethylene)acetoacetamide in c, theidentical process gives 0.3 g of3-methyl-4-(pyrid-2-aminocarbonyl)pyrazole as an off-white solid.

f. 3-Cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole.

By substituting cyclopropylnitromethane (Williams et al (1965) J. Org.Chem. 30: 2674-2675) for nitroethane in a, the identical process gives1.2 g of3-cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]-isoxazole as anoff white solid.

g. 3-Cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole.

Methyl cyclopropylcarbonylacetate (1.4 g) and 1.6 g of4-(trifluoromethyl)aniline are heated to 160° C. overnight. When thecrude product (or the product purified by crystallization or silica gelchromatography) is substituted forN-[4-trifluoromethylphenyl]acetoacetamide in the second step of b, theidentical process gives N-[4-trifluoromethylphenyl]-2-(ethoxymethylene)cyclopropylcarbonylacetamide. WhenN-[4-trifluoromethylphenyl]-2-(ethoxymethylene)cyclopropylcarbonyl-acetamide is substituted forN-[4-(trifluoromethyl)phenyl]-2-(ethoxymethylene)-acetoacetamide in c,the identical process gives3-cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]-pyrazole.

h. 3-Cyclopropyl-4-(pyrid-2-aminocarbonyl)isoxazole.

When 2-aminopyridine is substituted for 4-(trifluoromethyl)aniline in f,the identical process gives3-cyclopropyl-4-(pyrid-2-aminocarbonyl)isoxazole.

i. 3-Cyclopropyl-4-(pyrid-2-aminocarbonyl) pyrazole.

When 2-aminopyridine is substituted for 4-(trifluoromethyl)aniline in g,the identical process gives3-cyclopropyl-4-(pyrid-2-aminocarbonyl)pyrazole.

j.3-Methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3-Methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

By substituting 3-methyl-4(trifluoromethyl)aniline (Tordeaux, M., et al(1990) J. Chem. Soc. Perkin Trans. EN 8: 2293-2299) and4-(trifluoromethylsulfonyl)aniline (Jagupolski, M. (1954) Zh. Obshch.Khim. 24: 887-893, Engl. Ausg. S. 885-889) for4-(trifluoromethyl)aniline in the identical process of a,3-methyl-4-[3-methyl-4-(trifluoromethyl)-phenylaminocarbonyl]isoxazoleand 4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole areprepared.

k. 3-Methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

3-Methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

By substituting the appropriate amine for 4-(trifluoromethyl)aniline inthe identical process of g,3-methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]-pyrazoleand 3-Methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazoleare prepared.

l. 3-Allyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3-Allyl-4-(pyrid-2-aminocarbonyl)isoxazole;

3-Allyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3-Allyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

By substituting 4-nitro-1-butene (Seebach, D., et al (1978) Angew. Chem.GE 90: 479-480) for nitroethane in a, and then the appropriate aminesfor 4-(trifluoromethyl)-aniline in a, the identical process gives3-allyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole,3-allyl-4-(pyrid-2-aminocarbonyl)isoxazole,3-allyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole and3-allyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

m. 3-Allyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

3-Allyl-4-(pyrid-2-aminocarbonyl)pyrazole;

3-Allyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

3-Allyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

By substituting methyl 3-butenoate for methyl cyclopropylcarbonylacetatein g, and then the appropriate amines for 4-(trifluoromethyl)aniline ing, the identical process gives3-allyl-4-[4-(trifluoromethyl)phenylamino-carbonyl]pyrazole,3-allyl-4-(pyrid-2-aminocarbonyl)pyrazole),3-allyl-4-[3-methyl-4-(trifluoromethyl)phenyl-aminocarbonyl]pyrazole,and 3-allyl-4-[4-(trifluoro-methylsulfonyl)phenylaminocarbonyl]pyrazole.

n. 3,5-Dimethyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3,5-Dimethyl-4-(pyrid-2-aminocarbonyl)isoxazole;

3,5-Dimethyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3,5-Dimethyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]-isoxazole.

By using 3,5-dimethyl-4-chlorocarbonylisoxazole or by substituting ethylmethylpropiolate for ethyl propiolate in a and then the appropriateamines for 4-(trifluoromethyl)aniline in a, the identical process gives3,5-dimethyl-4-[4-(trifluoromethyl) phenylaminocarbonyl]isoxazole ,3,5-dimethyl-4-(pyrid-2-aminocarbonyl)isoxazole,3,5-dimethyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole(Example 25), and 3,5-dimethyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

o.3,5-Dimethyl-4-[4-(trifluoromethyl)phenylaminophenylaminocarbonyl]pyrazole;

3,5-Dimethyl-4-(pyrid-2-aminocarbonyl)isoxazole;3,5-Dimethyl-4-(pyrid-2-aminocarbonyl)pyrazole;

3,5-Dimethyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

3,5-Dimethyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

By substituting triethylorthoacetate for triethylorthoformate in thesecond step of b, and then substituting the appropriate amines for4-(trifluoromethyl)aniline in b, the identical process gives thecorresponding substituted 2-(ethoxymethylene)acetoacetamides. Bysubstituting the corresponding substituted 2-(ethoxymethylene)acetoacetamides for N-[4-(trifluoromethyl)phenyl]-2-(ethoxymethylene)acetoacetamide in c, the identical process gives3,5-dimethyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole,3,5-dimethyl-4-(pyrid-2-aminocarbonyl)pyrazole,3,5-dimethyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole,and3,5-dimethyl-4-[4-(trifluoromethyl-sulfonyl)phenylaminocarbonyl]pyrazole.

p.5-(2-Chlorophenyl)-3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

5-(2-chlorophenyl)-3-methyl-4-(pyrid-2-aminocarbonyl)isoxazole;

5-(2-chlorophenyl)-3-methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

5-(2-Chlorophenyl)-3-methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

By substituting ethyl 2-chlorophenylpropiolate (Newman, M. (1955) J.Amer. Chem. Soc. 77:5549) for ethyl propiolate in a, and then theappropriate amines for 4-(trifluoromethyl)aniline in a, the identicalprocess gives5-(2-chlorophenyl)-3-methyl-4-[4-(trifluoromethyl)phenylamino-carbonyl]isoxazole,5-(2-chlorophenyl)-3-methyl-4-(pyrid-2-aminocarbonyl)isoxazole,5-(2-chlorophenyl)-3-methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole,5-(2-chlorophenyl)-3-methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

q.5-(2-Chlorophenyl)-3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

5-(2-chlorophenyl)-3-methyl-4-(pyrid-2-aminocarbonyl) pyrazole;

5-(2-chlorophenyl)-3-methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

5-(2-Chlorophenyl)-3-methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

By substituting methyl 2-chlorobenzoylacetate formethylcyclopropylcarbonylacetate in g, and then substituting theappropriate amines for 4-(trifluoromethyl)aniline in g, the identicalprocess with triethylorthoacetate substituted for triethylorthoformatein b, gives5-(2-chlorophenyl)-3-methyl-4-[4-(trifluoromethyl)-phenylaminocarbonyl]prazole,5-(2-chlorophenyl)-3-methyl-4-(pyrid-2-aminocarbonyl)pyrazole,5-(2-chlorophenyl)-3-methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazoleand 5-(2-chlorophenyl)-3-methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

r.5-(2-Chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

5-(2-Chlorophenyl)-3-cyclopropyl-4-(pyrid-2-minocarbonyl)isoxazole;5-(2-Chlorophenyl)-3-cyclopropyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;5-(2-chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

By substituting ethyl 2-chlorophenylpropiolate for ethyl propiolate andcyclopropylnitromethane for nitroethane in a, and then the appropriateamines for 4-(trifluoromethyl)aniline in a, the identical process gives5-(2-chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole,5-(2-chlorophenyl)-3-cyclopropyl-4-(pyrid-2-aminocarbonyl)-isoxazole,and5-(2-chlorophenyl)-3-cyclopropyl-4-[3-methyl-4-(trifluoromethyl)phenylamino-carbonyl]isoxazoleand5-(2-chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

s.5-(2-Chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]-pyrazole;

5-(2-chlorophenyl)-3-cyclopropyl-4-(pyrid-2-aminocarbonyl)pyrazole;

5-(2-Chlorophenyl)-3-cyclopropyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

5-(2-chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

By substituting the appropriate amines for 4-(trifluoromethyl)aniline ing, and then by substituting triethyl-2-chloroorthobenzoate fortriethylorthoformate in b, the identical process of g gives5-(2-chlorophenyl)-3-cyclopropyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole,5-(2-chlorophenyl)-3-cyclopropyl-4-(pyrid-2-aminocarbonyl)pyrazole, and5-(2-chlorophenyl)-3-cyclopropyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazoleand5-(2-chlorophenyl)-3-cyclopropyl-4-[3-methyl-4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

t. 3-(2-Carboxyethyl)-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3-(2-carboxy-ethyl)-4-(pyrid-2-aminocarbonyl)isoxazole;

3-(2-Carboxyethyl)-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole;

3-(2-Carboxyethyl)-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole.

By substituting ethyl 4-(t-butoxycarbonylethyl) nitrobutane (from4-nitrobutyric acid methyl ester, Bissell, E. R. et al., Tetrahedron(1970), p 5737-5743) for ethyl propiolate in a, and then the appropriateamines for 4-(trifluoromethyl)aniline in a, the identical processfollowed by removal of the t-butyl group gives3-(2-carboxyethyl)-4-[4-(trifluoromethyl)phenylaminocarbonyl]isoxazole,3-(2-carboxyethyl)-4-(pyrid-2-aminocarbonyl)isoxazole,3-(2-carboxyethyl)-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]isoxazole,and3-(2-carboxyethyl)-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]isoxazole

u.3-(2-Carboxyethyl)-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

3-(2-carboxyethyl)-4-(pyrid-2-aminocarbonyl)pyrazole;

3-(2-Carboxyethyl)-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole;

3-(2-carboxyethyl)-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]pyrazole.

By substituting ethyl 2-(t-butoxycarbonyl)propionoacetate for ethylcyclopropylcarbonylacetate in g, and then substituting the appropriateamines for 4-(trifluoromethyl)aniline in g, the identical process,followed by removal of the t-butyl group, gives3-(2-carboxy-ethyl)-4-[4-(trifluoromethyl)phenylaminocarbonyl]pyrazole,3-(2-carboxyethyl)-4-(pyrid-2-aminocarbonyl)pyrazole,3-(2-carboxyethyl)-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]pyrazole,and 3-(2-carboxyethyl)-[4-(trifluoromethylsulfonyl)-phenylaminocarbonyl]pyrazole.

v. 4-(1,3-Benzodioxan-6-aminocarbonyl)-3-methyl-isoxazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-3-cyclopropylisoxazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-3,5-dimethyl-isoxazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-3-cyclopropyl-5-methylisoxazole;

4-(1,3-benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-methylisoxazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylisoxazole.

By substituting 6-amino-1,3-benzodioxane for 4-(trifluoro methyl)anilinein a, and then substituting the appropriate substituted priopiolates formethyl priopiolate and/or the appropriate substituted nitromethanes fornitroethane in a, the identical process gives4-(1,3-benzodioxan-6-aminocarbonyl)-3-methylisoxazole,4-(1,3-benzo-dioxan-6-aminocarbonyl)-3-cyclopropylisoxazole,4-(1,3-benzodioxan-6-aminocarbonyl)-3,5-dimethylisoxazole,4-(1,3-benzodioxan-6-aminocarbonyl)-3-cyclopropyl-5-methylisoxazole,4-(1,3-benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-methylisoxazole,and4-(1,3-benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylisoxazole.

w. 4-(1,3-Benzodioxan-6-aminocarbonyl)-3-methylpyrazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-3-cyclopropylpyrazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-3,5-dimethylpyrazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-3-cyclopropyl-5-methylpyrazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-methylpyrazole;

4-(1,3-Benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylpyrazole.

By substituting 6-amino-1, 3-benzodioxane for 4-(trifluoromethyl)anilinein g, and then the appropriate substituted acetoacetate for methylcyclopropylcarbonylacetoacetate and/or the appropriate substitutedorthoester for triethylorthoformate in the second step of b, theidentical process of the second step of b and of g gives4-(1,3-benzodioxan-6-aminocarbonyl)-3-methylpyrazole,4-(1,3-benzodioxan-6-aminocarbonyl)-3-cyclopropylpyrazole,4-(1,3-benzodioxan-6-aminocarbonyl)-3,5-dimethylpyrazole,4-(1,3-benzodioxan-6-aminocarbonyl)-3-cyclopropyl-5-methylpyrazole,4-(1,3-benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-methylpyrazole,and4-(1,3-benzodioxan-6-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylpyrazole.

x. 4-(1,3-Benzodioxol-5-aminocarbonyl)-3-methyl-isoxazole;

4-(1,3-Benzodioxol-5-aminocarbonyl)-3-cyclopropylisoxazole;

4-(1,3-Benzodioxol-5-aminocarbonyl])-3,5-dimethylisoxazole;

4-(1,3-Benzodioxol-5-aminocarbonyl)-3-cyclopropyl-5-methylisoxazole;

4-(1,3-Benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-methylisoxazole;

4-(1,3-benzo-dioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylisoxazole.

By substituting 5-amino-1,3-benzodioxole for 4-(trifluoromethyl)anilinein a, and then substituting the appropriate substituted propiolic esterfor ethyl propiolate and/or the appropriate stubstituted nitromethanefor nitroethane in a, the identical process gives4-(1,3-benzodioxol-5-aminocarbonyl)-3-methylisoxazole,4-(1,3-benzodioxol-5-aminocarbonyl)-3-cyclopropylisoxazole,4-(1,3-benzodioxol-5-aminocarbonyl])-3,5-dimethylisoxazole,4-(1,3-benzodioxol-5-aminocarbonyl)-3-cyclopropyl-5-methylisoxazole, and4-(1,3-benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-methylisoxazole,and4-(1,3-benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylisoxazole.

y. 4-(1,3-Benzodioxol-5-aminocarbonyl)-3-methyl-pyrazole;

4-(1,3-Benzodioxol-5-amino-carbonyl)-3-cyclopropylpyrazole;

4-(1,3-Benzodioxol-5-aminocarbonyl])-3,5-dimethylpyrazole;

4-(1,3-Benzodioxol-5-aminocarbonyl)-3-cyclopropyl-5-methylpyrazole;

4-(1,3-Benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-methylpyrazole;

4-(1,3-Benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylpyrazole.

By substituting 5-amino-1,3-benzodioxole for 4-(trifluoromethyl)anilinein g, and then substituting the appropriate substituted acetoacetate forethyl cyclopropylcarbonylacetate and/or the appropriate substitutedorthoformate for triethylorthoformate in the second step of b, theidentical process of the second step of b and of g gives4-(1,3-benzodioxol-5-aminocarbonyl)-3-methylpyrazole,4-(1,3-benzodioxol-5-aminocarbonyl)-3-cyclopropylpyrazole,4-(1,3-benzodioxol-5-aminocarbonyl])-3,5-dimethylpyrazole,4-(1,3-benzodioxol-5-aminocarbonyl)-3-cyclopropyl-5-methylpyrazole,4-(1,3-benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-methylpyrazole,and4-(1,3-benzodioxol-5-aminocarbonyl)-5-(2-chlorophenyl)-3-cyclopropylpyrazole.

z. 3-Methyl-4-[4-(trifluoro-methyl)phenylaminocarbonyl]thiazole;

3-Methyl-4-(pyrid-2-aminocarbonyl)thiazole;

3-Methyl-4-[3-methyl-4-trifluoromethyl)phenylaminocarbonyl]thiazole;

3-Methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]thiazole.

By substituting 3-methyl-4-thiazole carboxylic acid (Buttimore, D. etal.; J. Chem. Soc. (1963) p 2032-2039) for 3-methyl-4-isoxazolecarboxylic acid in a, and then substituting the appropriate amines for4-(trifluoromethyl)aniline in a, the identical process gives3-methyl-4-[4-(trifluoromethyl)phenylaminocarbonyl]thiazole,3-methyl-4-(pyrid-2-aminocarbonyl)thiazole,3-methyl-4-[3-methyl-4-(trifluoromethyl)phenylaminocarbonyl]thiazole,and 3-methyl-4-[4-(trifluoromethylsulfonyl)phenylaminocarbonyl]thiazole.

5. BRIEF DESCRIPTION OF THE TABLES

Table 1 is a comparison of the activity of leflunomide, its metaboliteand a compound of this invention as inhibitor of FGF induced DNAsynthesis alone or with added uridine.

Table 2 shows the results of the ability of several of the compounds ofthis invention to inhibit DNA synthesis induced by FGF, PDGF and EGF.

Table 3 shows the results of a subcutaneous xenograft experiment testingthe ability of Cmpd. 1 to inhibit tumor growth in vivo and, in addition,testing the toxic effects of Cmpd. 1 at the dose used.

6. BIOLOGICAL EVALUATION

It will be appreciated that, in any given series of compounds, aspectrum of biological activity will be afforded. In its most preferredembodiments, this invention relates to novel heteroarylcarboxamidesdemonstrating the ability to modulate PTK activity. The following assaysare employed to select those compounds demonstrating the optimal degreeof the desired activity.

As used herein, the phrase “optimal degree of the desired activity”refers to the lowest IC50, defined elsewhere herein, against a PTKrelated to a particular disorder so as to provide an organism,preferably a human, with a therapeutically effective amount of acompound of this invention at the lowest possible dosage.

The following in vitro assays may be used to determine the level ofactivity and effect of the different compounds of the present inventionon one or more of the RTKs. Similar assays can be designed along thesame lines for any PTK using techniques well known in the art.

The cellular/catalytic assays described herein are performed in an ELISAformat. The general procedure is a follows: a compound is introduced tocells expressing the test kinase, either naturally or recombinantly, forsome period of time after which, if the test kinase is a receptor, aligand known to activate the receptor's activity is added. The cells arelysed and the lysate is transferred to the wells of an ELISA platepreviously coated with a specific antibody recognizing the substrate ofthe enzymatic phosphorylation reaction. Non-substrate components of thecell lysate are washed away and the amount of phosphorylation on thesubstrate is detected with an antibody specifically recognizingphosphotyrosine compared with control cells that were not contacted witha test compound.

The cellular/biologic assays described herein measure the amount of DNAmade in response to activation of a test kinase, which is a generalmeasure of a proliferative response. The general procedure for thisassay is as follows: a compound is introduced to cells expressing thetest kinase, either naturally or recombinantly, for some period of timeafter which, if the test kinase is a receptor, a ligand known toactivate the receptor's activity is added. After incubation at leastovernight, a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or3H-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

1. Cellular/Catalytic Assays

Enzyme linked immunosorbent assays (ELISA) may be used to detect andmeasure the presence of PTK activity. The ELISA may be conductedaccording to known protocols which are described in, for example,Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual ofClinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am.Soc. Of Microbiology, Washington, D.C.

The disclosed protocol may be adapted for determining activity withrespect to a specific RTK. For example, the preferred protocols forconducting the ELISA experiments for specific RTKs is provided below.Adaptation of these protocols for determining a compound's activity forother members of the RTK family, as well as for CTKS, is well within thescope of knowledge of those skilled in the art.

a. FLK-1

An ELISA assay can be conducted to measure the kinase activity of theFLK-1 receptor and more specifically, the inhibition or activation of TKactivity on the FLK-1 receptor. Specifically, the following assay can beconducted to measure kinase activity of the FLK-1 receptor in cellsgenetically engineered to express Flk-1.

Materials And Methods.

Materials. The following reagents and supplies are being used:

a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);

b. Cappel goat anti-rabbit IgG (catalog no. 55641);

c. PBS (Gibco Catalog No. 450-1300EB);

d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20);

e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.);

f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% TritonX-100, and 10% glycerol);

g. EDTA (0.5 M (pH 7.0) as a 100× stock);

h. Sodium ortho vanadate (0.5 M as a 100× stock);

i. Sodium pyro phosphate (0.2M as a 100× stock);

j. NUNC 96 well V bottom polypropylene plates (Applied ScientificCatalog No. AS-72092);

k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);

l. DMEM with 1× high glucose L Glutamine (catalog No. 11965-050);

m. FBS, Gibco (catalog no. 16000-028);

n. L-glutamine, Gibco (catalog no. 25030-016);

o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 μg/100 μl stockin Milli-Q dH₂O and stored at −20° C.;

p. Affinity purified anti-FLK-1 antiserum;

q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley,et al., 1990, Cancer Research 50:1550-1558);

r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);

s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution(100 mM citric acid (anhydrous), 250 mM Na₂HPO₄ (pH 4.0), 0.5 mg/ml ABTS(Sigma catalog no. A-1888)), solution should be stored in dark at 4° C.until ready for use;

t. H₂O₂ (30% solution) (Fisher catalog no. H325);

u. ABTS/H₂O₂ (15 ml ABTS solution, 2 μl H₂O₂) prepared 5 minutes beforeuse and left at room temperature;

v. 0.2 M HCl stock in H₂O;

w. dimethylsulfoxide (100%)(Sigma Catalog No. D-8418); and

y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).

Protocol. The following protocol is being used for conducting the assay:

1. Coat Corning 96-well elisa plates with 1.0 μg per well CappelAnti-rabbit IgG antibody in 0.1M Na₂CO₃ pH 9.6. Bring final volume to150 μl per well. Coat plates overnight at 4° C. Plates can be kept up totwo weeks when stored at 4° C.

2. Grow cells in Growth media(DMEM, supplemental with 2.0 mML-Glutamine, 10% FBS) in suitable culture dishes until confluent at 37°C., 5% CO₂.

3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene96-well roundbottom cell plates, 25.000 cells/well in 200 μl of growthmedia.

4. Grow cells at least one day at 37° C., 5% CO₂.

5. Wash cells with D-PBS 1×.

6. Add 200 μl/well of starvation media (DMEM, 2.0 mM l-Glutamine, 0.1%FBS). Incubate overnight at 37° C., 5% CO₂.

7. Dilute Compounds 1:20 in polypropylene 96 well plates usingstarvation media. Dilute dimethylsulfoxide 1:20 for use in controlwells.

8. Remove starvation media from 96 well cell culture plates and add 162μl of fresh starvation media to each well.

9. Add 18 μl of 1:20 diluted Compound dilution (from step 7) to eachwell plus the 1:20 dimethylsulfoxide dilution to the control wells(+/−VEGF), for a final dilution of 1:200 after cell stimulation. Finaldimethylsulfoxide is 0.5%. Incubate the plate at 37° C., 5% CO₂ for twohours.

10. Remove unbound antibody from ELISA plates by inverting plate toremove liquid. Wash 3 times with TBSW+0.5% ethanolamine, pH 7.0. Pat theplate on a paper towel to remove excess liquid and bubbles.

11. Block plates with TBSW+0.5% Ethanolamine, pH 7.0, 150 μl per well.Incubate plate thirty minutes while shaking on a microtiter plateshaker.

12. Wash plate 3 times as described in step 10.

13. Add 0.5 μg/well affinity purified anti-FLU-1 polyclonal rabbitantiserum. Bring final volume to 150 μl/well with TBSW+0.5% ethanolaminepH 7.0. Incubate plate for thirty minutes while shaking.

14. Add 180 μl starvation medium to the cells and stimulate cells with20 μl/well 10.0 mM sodium ortho vanadate and 500 ng/ml VEGF (resultingin a final concentration of 1.0 mM sodium orthovanadate and 50 ng/mlVEGF per well) for eight minutes at 37° C., 5% CO₂. Negative controlwells receive only starvation medium.

15. After eight minutes, media should be removed from the cells andwashed one time with 200 μl/well PBS.

16. Lyse cells in 150 μl/well HNTG while shaking at room temperature forfive minutes. HNTG formulation includes sodium ortho vanadate, sodiumpyro phosphate and EDTA.

17. Wash ELISA plate three times as described in step 10.

18. Transfer cell lysates from the cell plate to elisa plate andincubate while shaking for two hours. To transfer cell lysate pipette upand down while scrapping the wells.

19. Wash plate three times as described in step 10.

20. Incubate ELISA plate with 0.02 μg/well UB40 in TBSW+05%ethanolamine. Bring final volume to 150 μl/well. Incubate while shakingfor 30 minutes.

21. Wash plate three times as described in step 10.

22. Incubate ELISA plate with 1:10,000 diluted EIA grade goat anti-mouseIgG conjugated horseradish peroxidase in TBSW+0.5% ethanolamine, pH 7.0.Bring final volume to 150 μl/well. Incubate while shaking for thirtyminutes.

23. Wash plate as described in step 10.

24. Add 100 μl of ABTS/H₂O₂ solution to well. Incubate ten minutes whileshaking.

25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final to stop the colordevelopment reaction. Shake 1 minute at room temperature. Remove bubbleswith slow stream of air and read the ELISA plate in an ELISA platereader at 410 nm.

b. HER-2 ELISA

Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells.

HER2 kinase activity in whole EGFR-NIH3T3 cells are measured asdescribed below:

Materials and Reagents. The following materials and reagents are beingused to conduct the assay:

a. EGF: stock concentration=16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.

b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellulardomain).

c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see, Fendley,et al., supra).

d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidaseconjugate, TAGO, Inc., Burlingame, Calif.

e. TBST buffer:

Tris-HCl, pH 7.2 50 mM NaCl 150 mM Triton X-100 0.1

f. HNTG 5× stock:

HEPES 0.1 M NaCl 0.75 M Glycerol 50% Triton X-100 1.0%

g. ABTS stock:

Citric Acid 100 mM Na₂HPO₄ 250 mM HCl, conc. 0.5 pM ABTS* 0.5 mg/ml*(2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)).

Keep solution in dark at 4° C. until use.

h. Stock reagents of:

EDTA 100 mM pH 7.0

Na₃VO₄ 0.5 M

Na₄(P₂O₇) 0.2 M

Procedure. The following protocol is being used:

A. Pre-coat ELISA Plate

1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101antibody at 0.5 g per well in PBS, 100 μl final volume/well, and storeovernight at 4° C. Coated plates are good for up to 10 days when storedat 4° C.

2. On day of use, remove coating buffer and replace with 100 μl blockingbuffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubate theplate, shaking, at room temperature (about 23° C. to 25° C.) for 30minutes. Just prior to use, remove blocking buffer and wash plate 4times with TBST buffer.

B. Seeding Cells

1. An NIH3T3 cell line overexpressing a chimeric receptor containing theEGFR extracellular domain and intracellular HER2 kinase domain can beused for this assay.

2. Choose dishes having 80-90% confluence for the experiment. Trypsinizecells and stop reaction by adding 10% fetal bovine serum. Suspend cellsin DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, atroom temperature for 5 minutes.

3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), andcount the cells using trypan blue. Viability above 90% is acceptable.Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000cells per well, 100 μl per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO₂ at 37° C. for about 40 hours.

C. Assay Procedures

1. Check seeded cells for contamination using an inverted microscope.Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer5 l to a TBST well for a final drug dilution of 1:200 and a final DMSOconcentration of 1%. Control wells receive DMSO alone. Incubate in 5%CO₂ at 37° C. for two hours.

2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of10 μl dilute EGF (1:12 dilution), 100 nM final concentration isattained.

3. Prepare fresh HNTG* sufficient for 100 μl per well; and place on ice.

HNTG* (10 ml): 2.0 ml HNTG stock milli-Q H₂0 7.3 ml EDTA, 100 mM, pH 7.00.5 ml Na₃VO₄, 0.5 M 0.1 ml Na₄(P₂O₇), 0.2 M 0.1 ml

4. After 120 minutes incubation with drug, add prepared SGF ligand tocells, 10 μl per well, to a final concentration of 100 nM. Control wellsreceive DMEM alone. Incubate, shaking, at room temperature, for 5minutes.

5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG*to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, removeblocking buffer from other ELISA plate and wash with TBST as describedabove.

6. With a pipette tip securely fitted to a micropipettor, scrape cellsfrom plate and homogenize cell material by repeatedly aspirating anddispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked,and washed ELISA plate. Incubate shaking at room temperature for onehour.

7. Remove lysate and wash 4 times with TBST. Transfer freshly dilutedanti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shakingat room temperature for 30 minutes in the presence of the anti-Ptyrantiserum (1:3000 dilution in TBST).

8. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transferthe freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at100 μl per well. Incubate shaking at room temperature for 30 minutes(anti-rabbit IgG antibody: 1:3000 dilution in TBST).

9. Remove TAGO detection antibody and wash 4 times with TBST. Transferfreshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl per well.Incubate shaking at room temperature for 20 minutes. (ABTS/H₂O₂solution: 1.0 μl 30% H₂O₂ in 10 ml ABTS stock).

10. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), and determineO.D. at 410 nm.

11. The maximal phosphotyrosine signal is determined by subtracting thevalue of the negative controls from the positive controls. The percentinhibition of phosphotyrosine content for extract-containing wells isthen calculated, after subtraction of the negative controls.

c. PDGF-R ELISA

All cell culture media, glutamine, and fetal bovine serum can bepurchased from Gibco Life Technologies (Grand Island, N.Y.) unlessotherwise specified. All cells are grown in a humid atmosphere of 90-95%air and 5-10% CO₂ at 37° C. All cell lines are routinely subculturedtwice a week and are negative for mycoplasma as determined by theMycotect method (Gibco).

For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU)are grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA,1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in0.5% serum at 25,000 to 30,000 cells per well. After overnightincubation in 0.5% serum-containing medium, cells are changed toserum-free medium and treated with test compound for 2 hr in a 5% CO₂,37° C. incubator. Cells are then stimulated with ligand for 5-10 minutefollowed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5mM EDTA, 5 mM Na₃VO₄, 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates(0.5 mg/well in PBS) are transferred to ELISA plates previously coatedwith receptor-specific antibody and which had been blocked with 5% milkin TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100) atroom temperature for 30 min. Lysates are incubated with shaking for 1hour at room temperature. The plates are washed with TBST four times andthen incubated with polyclonal anti-phosphotyrosine antibody at roomtemperature for 30 minutes. Excess anti-phosphotyrosine antibody wasremoved by rinsing the plate with TBST four times. Goat anti-rabbit IgGantibody was added to the ELISA plate for 30 min at room temperaturefollowed by rinsing with TBST four more times. ABTS (100 mM citric acid,250 mM Na₂HPO₄ and 0.5 mg/mL2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H₂O₂ (1.2 mL30% H₂O₂ to 10 ml ABTS) was added to the ELISA plates to start colordevelopment. Absorbance at 410 nm with a reference wavelength of 630 nmwas recorded about 15 to 30 min after ABTS addition.

d. IGF-I RECEPTOR ELISA

The following protocol may be used to measure phosphotyrosine level onIGF-I receptor, which indicates IGF-I receptor tyrosine kinase activity.

Materials And Reagents. The following materials and reagents are used:

a. The cell line used in this assay is 3T3/IGF-1R, a cell linegenetically engineered to overexpresses IGF-1 receptor.

b. NIH3T3/IGF-1R is grown in an incubator with 5% CO₂ at 37° C. Thegrowth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.

c. Affinity purified anti-IGF-1R antibody 17-69.

d. D-PBS:

KH₂PO₄ 0.20 g/l K₂HPO₄ 2.16 g/l KCl 0.20 g/1 NaCl 8.00 g/l (pH 7.2)

e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat DryMilk).

f. TBST buffer:

Tris-HCl 50 mM NaCl 150 mM (pH 7.2/HCl 10N Triton X-100 0.1%

 Stock solution of TBS (10×) is prepared, and Triton X-100 is added tothe buffer during dilution.

g. HNTG buffer:

HEPES 20 mM NaCl 150 mM (pH 7.2/HCl 1N) Glycerol 10% Triton X-100 0.2%

 Stock solution (5×) is prepared and kept at 4° C.

h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) as 100× stock.

I. Na₃VO₄: 0.5 M as 100× stock and aliquots are kept in −80° C.

j. Na₄P₂O₇: 0.2 M as 100× stock.

k. Insulin-like growth factor-1 from Promega (Cat#G5111).

l. Rabbit polyclonal anti-phosphotyrosine antiserum.

m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat.No. 4520, Lot No. 1802): Tago, Inc., Burlingame, Calif.

n. ABTS (2.2′-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution:

Citric acid 100 mM Na₂HPO₄ 250 mM (pH 4.0/1N HCl) ABTS 0.5 mg/ml

 ABTS solution should be kept in dark and 4° C. The solution should bediscarded when it turns green.

o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4° C.

Procedure. All the following steps are conducted at room temperatureunless it is specifically indicated. All ELISA plate washings areperformed by rinsing the plate with tap water three times, followed byone TBST rinse. Pat plate dry with paper towels.

A. Cell Seeding:

1. The cells, grown in tissue culture dish (Corning 25020-100) to 80-90%confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100,GIBCO).

2. Resuspend the cells in fresh DMEM+10% FBS+2 mM L-Glutamine, andtransfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000cells/well (100 μl/well). Incubate for 1 day then replace medium toserum-free medium (90/μl) and incubate in 5% CO₂ and 37° C. overnight.

B. ELISA Plate Coating and Blocking:

1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at0.5 μg/well in 100 μl PBS at least 2 hours.

2. Remove the coating solution, and replace with 100 μl Blocking Buffer,and shake for 30 minutes. Remove the blocking buffer and wash the platejust before adding lysate.

C. Assay Procedures:

1. The drugs are tested in serum-free condition.

2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-wellpoly-propylene plate, and transfer 10 μl/well of this solution to thecells to achieve final drug dilution 1:100, and final DMSO concentrationof 1.0%. Incubate the cells. in 5% CO₂ at 37° C. for 2 hours.

3. Prepare fresh cell lysis buffer (HNTG*)

HNTG   2 ml EDTA 0.1 ml Na₃VO₄ 0.1 ml Na₄(P₂O₇) 0.1 ml H₂0 7.3 ml

4. After drug incubation for two hours, transfer 10 μl/well of 200 nMIGF-1 Ligand in PBS to the cells (Final Conc.=20 nM), and incubate at 5%CO₂ at 37° C. for 10 minutes.

5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Lookat cells under microscope to see if they are adequately lysed.

6. Use a 12-channel pipette to scrape the cells from the plate, andhomogenize the lysate by repeat aspiration and dispense. Transfer allthe lysate to the antibody coated ELISA plate, and shake for 1 hour.

7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 withTBST) 100 μl/well, and shake for 30 minutes.

8. Remove anti-pTyr, wash the plate, transfer Tago (1:3,000 with TBST)100 μl/well, and shake for 30 minutes.

9. Remove detection antibody, wash the plate, and transfer freshABTS/H202 (1.2 μl H₂O₂ to 10 ml ABTS) 100 μl/well to the plate to startcolor development.

10. Measure OD at 410 nm with a reference wavelength of 630 nm inDynatec MR5000.

e. EGF Receptor ELISA

EGF Receptor kinase activity in cells genetically engineered to expresshuman EGF-R is measured as described below:

Materials and Reagents. The following materials and reagents are used

a. EGF Ligand: stock concentration=16.5 μM; EGF 201, TOYOBO, Co., Ltd.Japan.

b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellulardomain).

c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).

d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidaseconjugate, TAGO, Inc., Burlingame, Calif.

e. TBST buffer:

Tris-HCl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1

f. HNTG 5× stock:

HEPES 0.1 M NaCl 0.75 M Glycerol 50 Triton X-100 1.0%

g. ABTS stock:

Citric Acid 100 mM Na₂HPO₄ 250 mM HCl, conc. 4.0 pH ABTS* 0.5 mg/ml

 Keep solution in dark at 4° C. until used.

h. Stock reagents of:

EDTA 100 mM pH 7.0

Na₃VO₄ 0.5 M

Na₄(P₂O₇) 0.2 M

Procedure. The following protocol was used:

A. Pre-coat ELISA Plate

1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101antibody at 0.5 μg per well in PBS, 150 μl final volume/well, and storeovernight at 4° C. Coated plates are good for up to 10 days when storedat 4° C.

2. On day of use, remove coating buffer and replace with blocking buffer(5% Carnation Instant NonFat Dry Milk in PBS). Incubate the plate,shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes.Just prior to use, remove blocking buffer and wash plate 4 times withTBST buffer.

B. Seeding Cells

1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can beuse for this assay.

2. Choose dishes having 80-90% confluence for the experiment. Trypsinizecells and stop reaction by adding 10% CS DMEM medium. Suspend cells inDMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm, andonce at room temperature for 5 minutes.

3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), andcount the cells using trypan blue. Viability above 90% is acceptable.Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000cells per well, 100 μl per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO₂ at 37° C. for about 40 hours.

C. Assay Procedures.

1. Check seeded cells for contamination using an inverted microscope.Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer5 μl to a test well for a final drug dilution of 1:200 and a final DMSOconcentration of 1%. Control wells receive DMSO alone. Incubate in 5%CO₂ at 37° C. for one hour.

2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of10 μl dilute EGF (1:12 dilution), 25 nM final concentration is attained.

3. Prepare fresh 10 ml HNTG* sufficient for 100 μl per well whereinHNTG* comprises: HNTG stock (2.0 ml), milli-Q H₂O (7.3 ml), EDTA, 100mM, pH 7.0 (0.5 ml), Na₃VO₄ 0.5 M (0.1 ml) and Na₄ (P₂O₇), 0.2 M (0.1ml).

4. Place on ice.

5. After two hours incubation with drug, add prepared EGF ligand tocells, 10 μl per well, to yield a final concentration of 25 nM. Controlwells receive DMEM alone. Incubate, shaking, at room temperature, for 5minutes.

6. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTGto cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, removeblocking buffer from other ELISA plate and wash with TBST as describedabove.

7. With a pipette tip securely fitted to a micropipettor, scrape cellsfrom plate and homogenize cell material by repeatedly aspirating anddispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked,and washed ELISA plate. Incubate shaking at room temperature for onehour.

8. Remove lysate and wash 4 times with TBST. Transfer freshly dilutedanti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shakingat room temperature for 30 minutes in the presence of the anti-Ptyrantiserum (1:3000 dilution in TBST).

9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transferthe freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plateat 100 μl per well. Incubate shaking at room temperature for 30 minutes(anti-rabbit IgG antibody: 1:3000 dilution in TBST).

10. Remove detection antibody and wash 4 times with TBST. Transferfreshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl per well.Incubate at room temperature for 20 minutes. ABTS/H₂O₂ solution: 1.2 μl30% H₂O₂ in 10 ml ABTS stock.

11. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), and determineO.D. at 410 nm.

12. The maximal phosphotyrosine signal is determined by subtracting thevalue of the negative controls from the positive controls. The percentinhibition of phosphotyrosine content for extract-containing wells isthen calculated, after subtraction of the negative controls.

2. Cellular/Biologic Assays

Assay 1: PDGF-Induced BrdU Incorporation Assay

Materials and Reagents:

(1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1× PBS, pH 7.4, made in house.

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human PDGF-R.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (PDGF=3.8 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (PDGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 2: EGF-Induced BrdU Incorporation Assay

Materials and Reagents

(1) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1× PBS, pH 7.4, made in house.

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human EGF-R.

Protocol

(1) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, ina 96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and testcompounds are added to the cells simultaneously. The negative controlwells receive serum free DMEM with 0.1% BSA only; the positive controlcells receive the ligand (EGF) but no test compound. Test compounds areprepared in serum free DMEM with ligand in a 96 well plate, and seriallydiluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 3: EGF-Induced Her2 -Driven BrdU Incorporation

Materials and Reagents:

(1) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1× PBS, pH 7.4, made in house.

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line engineered to express a chimeric receptor having theextra-cellular domain of EGF-R and the intra-cellular domain of Her2.

Protocol:

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96-well plate. Cells are incubated overnight at 37° in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and testcompounds are added to the cells simultaneously. The negative controlwells receive serum free DMEM with 0.1% BSA only; the positive controlcells receive the ligand (EGF) but no test compound. Test compounds areprepared in serum free DMEM with ligand in a 96 well plate, and seriallydiluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 4: IGF1-Induced BrdU incorporation Assay

Materials and Reagents:

(1) IGF1 Ligand: human, recombinant; G511, Promega Corp, USA.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1× PBS, pH 7.4, made in house.

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human IGF-1receptor.

Protocol:

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96-well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (IGF1) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 16 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

g. HUV-EC-C Assay

The following protocol may also be used to measure a compound's activityagainst PDGF-R, FGF-R or Flk-1/KDR, all of which are naturally expressedby HUV-EC cells.

DAY 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection; catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS; obtained from GibcoBRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company; catalogue no. C-1544).The 0.05% trypsin was made by diluting 0.25% trypsin/1 mM EDTA (Gibco;catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific; catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200×g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL; catalogue no. 21127-014) +0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter®v Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well; incubate ˜24 h at 37° C., 5% CO₂.

DAY 1

1. Make up two-fold drug titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of drugat 200 μM (4× the final well concentration) to the top well of aparticular plate column. Since the stock drug concentration is usually20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

Therefore, diluent made up to 2% DMSO in assay medium (F12K +0.5% fetalbovine serum) is used as diluent for the drug titrations in order todilute the drug but keep the DMSO concentration constant. Add thisdiluent to the remaining wells in the column at 60 μl/well. Take 60 μlfrom the 120 μl of 200 μM drug dilution in the top well of the columnand mix with the 60 μl in the second well of the column. Take 60 μl fromthis well and mix with the 60 μl in the third well of the column, and soon until two-fold titrations are completed. When the next-to-the-lastwell is mixed, take 60 μl of the 120 μl in this well and discard it.Leave the last well with 60 μl of DMSO/media diluent as anon-drug-containing control. Make 9 columns of titrated drug, enough fortriplicate wells each for 1) VEGF (obtained from Pepro Tech Inc.,catalogue no. 100-200, 2) endothelial cell growth factor (ECGF) (alsoknown as acidic fibroblast growth factor, or aFGF) (obtained fromBoehringer Mannheim Biochemica, catalogue no. 1439 600); or, 3) humanPDGF B/B (1276-956, Boehringer Mannheim, Germany) and assay mediacontrol. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the drug dilutions to the 96-well assay platescontaining the 0.8-1.0×10⁴ cells/100 μl/well of the HUV-EC-C cells fromday 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each drug condition. As with the drugs, the growthfactor concentrations are 4× the desired final concentration. Use theassay media from day 0 step 2 to make the concentrations of growthfactors. Incubate approximately 24 hours at 37° C., 5% CO₂. Each wellwill have 50 μl drug dilution, 50 μl growth factor or media, and 100 ulcells,=200 ul/well total. Thus the 4× concentrations of drugs and growthfactors become 1× once everything has been added to the wells.

DAY 2

1. Add ³H-thymidine (Amersham; catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media +10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI was obtained from Gibco BRL, catalogue no. 11875-051.

DAY 3

1. Freeze plates overnight at −20° C.

DAY 4

1. Thaw plates and harvest with a 96-well plate harvester (TomtecHarvester 96®) onto filter mats (Wallac; catalogue no. 1205-401); readcounts on a Wallac Betaplate™ liquid scintillation counter.

C. In Vivo Animal Models

1. Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619),A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung,ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435) and NIH 3T3fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-1R orany other test kinase.

The following protocol can be used to perform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students' t-test. Test compounds in 50-100 μL excipient (DMSO,or VPD:D5W) was delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

2. Tumor Invasion Model

The following tumor invasion model has been developed and maybe used forthe evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells was performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg) are administered intraperitoneally. A midline incision is doneto expose the abdominal cavity (approximately 1.5 cm in length) toinject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin was closed by using would clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases, to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurements of tumor size, grade of invasion,immunochemistry, and in situ hybridization).

D. EXAMPLES OF ASSAYS.

The following are examples of the results of specific assays used toevaluate the activity of the compounds of this invention. The assaysshown are exemplary only and are not to be construed as limiting in anymanner.

1. Inhibition of Ligand-Stimulated DNA Synthesis.

The following example illustrates the ability of the compounds of theinvention to inhibit FGFR-stimulated and PDGFR-stimulated DNA synthesisin cells. DNA synthesis is required for many of the activities of FGFRand PDGFR including, but not limited to, cell proliferation. Uridine isadded in one set of samples to overcome any contribution made byinhibition of DHOD and just evaluate the inhibition of PDGFR or FGFRsignaling. (See Greene, et al., Biochem. Pharmacol., 50(6):861 (1995),Nair, et al., Immunology Letters, 47:171 (1995)).

MATERIALS AND METHODS

(1) EGF: mouse EGF, 201; Toyobo,Co., Ltd. Japan; PDGF, BoehringerMannheim, Germany; FGF, Gibco.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4),Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1× phosphate buffered saline, pH 7.4

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) NIH3T3 clone C7 (3T3/EGFRc7)(Honegger et al., Cell 51:199-209,1987)) engineered to over-express human EGF receptor. These cellsnatively express FGFR and PDGFR.

PROTOCOL

(1) 3T3/EGFRc7 cells were seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gln in a 96 well plate. Cells are incubated overnight at 37 degreesC. in 5% CO₂.

(2) After 24 hours, the cells were washed with PBS, and then serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (2 nM EGF or 1.5 nM FGF or 3.8 nM PDGF) prepared inDMEM with 0.1% BSA and 30 μM (final concentration) uridine) and testcompound was added to the cells simultaneously. The negative controlwells received serum free DMEM with 0.1% BSA only; the positive controlcells received ligand but no test compound. Test compound was preparedin serum free DMEM with ligand in a 96 well plate, and serially dilutedfor 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) was added and the cells were incubated withBrdU (final concentration=10 μM) for 1.5 hours. (5) After incubationwith labeling reagent, the medium was removed by decanting and tappingthe inverted plate on a paper towel. FixDenat solution was added (50μl/well) and the plates incubated at room temperature for 45 minutes ona plate shaker.

(6) The FixDenat solution was thoroughly removed by decanting andtapping the inverted plate on a paper towel. Milk is added (5%dehydrated milk in PBS, 200 μl/well) as a blocking solution and theplate was incubated for 30 minutes at room temperature on a plateshaker.

(7) The blocking solution was removed by decanting and the wells werewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) was added (100 μl/well) and the plate was incubated for 90 minutesat room temperature on a plate shaker.

(8) The antibody conjugate was thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate dried by inverting andtapping on a paper towel.

(9) TMB substrate solution was added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color developmentwas sufficient for photometric detection.

(10) The absorbance of the samples was measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

RESULTS

In a first experiment, the activity of leflunomide, its metabolite and acompound of this invention are compared in their ability to inhibit FGFinduced DNA synthesis alone or with added uridine. As shown in Table 1below, the ability of the metabolite to inhibit DNA synthesis iscompletely abolished by the addition of uridine and that of leflunomidereduced, demonstrating that the inhibitory effect is due to inhibitionof DHOD, not inhibition of FGFR signaling. In contrast, the inhibitoryactivity of claimed compound in not decreased, and is even slightlyincreased, demonstrating that it is inhibiting FGFR signaling. Similarexperiments were conducted using PDGF stimulation with similar results.Also tested was the known DHOD inhibitor brequinar which inhibited DNAsynthesis in the absence of uridine (IC50=1.6 μM) and was inactive inthe presence of uridine (IC50=>100 μM).

TABLE 1 Compound IC50 − uridine IC50 + uridine leflunomide 20 μM 80 μMA771726 20 μM >100 μM  3-Methyl-4-[4- 85 μM 70 μM(trifluoromethyl)phenyl- aminocarbonyl]isoxazole (cmpd. 1)

In another experiment, several of the compounds of this invention weretested for their ability to inhibit DNA synthesis induced by FGF, PDGFand EGF. The results, shown in Table 2 below, indicate that thecompounds of the invention are selective for inhibition of FGF and PDGFinduced signaling compared to EGF signaling.

TABLE 2 Compound PDGF-induced FGF-induced EGF-induced 3-Methyl-4-[4- 95μM >100 μM >100 μM (trifluoromethyl)- phenylaminocarbonyl]- pyrazole(cmpd. 2) 3,5-Dimethyl-4-[4- >100 μM  >100 μM >100 μM (trifluoromethyl)-phenylaminocarbonyl]- isoxazole (cmpd. 21) 3-Methyl-4-[4- 90 μM  65μM >100 μM (trifluoromethyl)- phenylaminocarbonyl]- isoxazole (cmpd. 1)

2. Inhibition of Tumor Growth in vivo

The following example demonstrates the ability of Cmpd. 1 to inhibit thein vivo growth of tumors characterized by inappropriate FGFR and/orPDGFR activity. The FGFR expressing cells are from two glioblastomas(C6, ATCC CRL 107, Powell and Klagsbrun, Exp. Cell Res., 209:224 (1993);for PDGER, see Strawn, et al., J. Biol. Chem., 269:21215 (1995).

MATERIALS AND METHODS

Female athymic mice (BALB/c, nu/nu) were obtained from SimonsenLaboratories (Gilroy, Calif.). All animals were maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They received sterile rodent chow and water ad libitum.

Cell lines were grown in Ham's F10 plus 5% fetal bovine serum (FBS) and2 mM glutamine (GLN). All cell culture media, glutamine, and fetalbovine serum were purchased from Gibco Life Technologies (Grand Island,N.Y.) unless otherwise specified. All cells were grown in a humidatmosphere of 90-95% air and 5-10% CO₂ at 37° C. All cell lines wereroutinely subcultured twice a week and were negative for mycoplasma asdetermined by the Mycotect method (Gibco).

Cells were harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets were resuspended in sterile PBS ormedia (without FBS) and the cells were implanted into the hindflank ofthe mice (8-10 mice per group, 3×10⁶ cells/animal). Tumor growth wasmeasured over 3 weeks using venier calipers. Tumor volumes werecalculated as a product of length×width×height unless otherwiseindicated. P values were calculated using the Students' t-test. Cmpd. 1in 50 μL excipient (DMSO) was delivered by IP bolus injection daily.

RESULTS

The results of the subcutaneous xenograft experiment, shown in Table 3below, demonstrates that administration of Cmpd. 1 significantlyinhibited the tumor growth in vivo and had no toxic effect at the dosetested.

TABLE 3 inhibition % of Treatment Day control) mortality p-value DMSOalone 20 — 0 — 30 mg/kg/day 8 34 0 0.0158 (cmpd. 1) 10 42 0 0.0034 13 480 0.0520 15 47 0 0.0392 17 50 0 0.0188 20 52 0 0.0064

3. Inhibition of Tumor Growth and Metastasis in vivo.

The following example can be used to test the ability of the compoundsof the invention to inhibit growth and metastasis of a tumor cell lineexpressing FGFR and PDGFR (C6 cells).

MATERIALS AND METHODS

Ten to 12 week old athymic Balb/c nu/nu mice are obtained from SimonsenLaboratory (Gilroy, Calif.) and maintained in a pathogen-freeenvironment throughout the experiments.

C6 cells (ATCC CCL 107) are grown and maintained in F-10 medium (LifeTechnologies, Inc. Grand Island, N.Y.) supplemented with 10% fetalbovine serum, 2 mM glutamine in a 5% CO2 environment. Approximately 80%confluent cultures are harvested by brief trypsinization (0.0625%trypsin-0.25 mM EDTA in Cell Dissociation Medium) (Life Technologies)and resuspended at a final concentration of 8×10⁷ cells per ml inmagnesium and calcium free phosphate buffered saline for implantation.Cell viability is determined by Trypan blue exclusion and found to be>95%.

On the day of implantation, animals are anesthetized with eitherisoflurane or Ketaset and Rompun and the abdomen is prepared for sterilesurgery. A small abdominal incision is made and the ascending colonidentified. The gut is then placed on strips of sterile gauze beforeinjection. Two million viable tumor cells in 0.025 ml PBS are injectedunder the serosa into the muscularis/submuscularis by means of a steriletuberculin syringe and a 27 gauge needle. Cells are injected so as tovisibly infiltrate between the submucosal and subserosal tissues. Theserosal surface at the injection site is dabbed gently with 70%isopropyl alcohol pads to kill tumor cells that may have escaped. Theorgans are replace in situ. The abdominal wall is closed with continuousnylon sutures. The outer skin is then closed using wound clips which areremoved seven days post implantation.

To ensure that cell implantation is properly performed, after 7 days,several control animals are euthanized by cerebral dislocation , theabdominal organs and thorax examined for the presence of macroscopic“primary” colonic tumors and metastases. Pilot studies demonstrated thatat this time intracolonic tumors of approximately 5 to 7 mm³ are presentwithout peritoneal spread of tumor after injection of C6 cells.

One day following implantation of cells, animals are treated once dailyintraperitoneally with either MCTA at 20 mg/kg/day in VPD:D5W or vehiclealone in a 0.1 ml bolus. The health of the animals is monitored dailyand if signs of severe discomfort or pain is observed or the animal isdeemed to be moribund, animals are sacrificed humanely. Dosing of theanimals continued until all surviving animals in the experiment aredeemed moribund. When possible, the local tumor growth on the colon ismeasured and the major organs such as lung, heart, spleen, liver andkidney are resected from moribund animals and submitted forhistopathological analysis.

E. MEASUREMENT OF CELL TOXICITY

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index: IC₅₀/LD₅₀. IC₅₀, the dose required toachieve 50% inhibition, can be measured using standard techniques suchas those described herein. LD₅₀, the dosage which results in 50%toxicity, can also be measured by standard techniques (Mossman, 1983, J.Immunol. Methods, 65:55-63), by measuring the amount of LDH released(Korzeniewski and Callewaert, 1983, J. Immunol. Methods, 64:313; Deckerand Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by measuringthe lethal dose in animal models. Compounds with a large therapeuticindex are preferred. The therapeutic index should be greater than 2,preferably at least 10, more preferably at least 50.

CONCLUSION

Thus, it will be appreciated that the compounds, methods andpharmacological compositions of the present invention modulate PTKactivity and therefore to be effective as therapeutic agents againstPTK-related disorders.

Although certain embodiments and examples have been used to describe thepresent invention, it will be apparent to those skilled in the art thatchanges to the embodiments and examples shown may be made withoutdeparting from the scope and spirit of the invention.

Other embodiments are within the following claims.

What is claimed:
 1. An isoxazole-4-carboxamide compound having thefollowing chemical structure:

wherein: R¹ is selected from the group consisting of alkyl, cycloalkyl,alkenyl, alkynyl, and heteroalicyclic; R³ is selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl and heteroalicyclic; Z is selected from the group consistingof oxygen and sulfur; R⁴ is selected from the group consisting ofhydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl heteroaryl,heteroalicyclic, sulfonyl, trihalomethanesulfonyl, hydroxy, alkoxy andC-carboxy; R⁵, R⁶, R⁷, R⁸ and R⁹ are independently selected from thegroup consisting of hydrogen, alkyl, trihaloalkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,cycloalkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy,thioalkyoxy, thiocycloalkoxy, thioheteraryloxy, thioheteralicycloxy,cyano, C—O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, silyl,phosphonyl, C-carboxy, O-carboxy, N-amido, C-amido, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethanesulfonyl, guanyl, guanidino,trihalomethanesulfonamido, amino and —NR¹³R¹⁴; wherein, R¹³ and R¹⁴ areindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, carbonyl, C-carboxy, sulfonyl, trihalomethanesulfonyland, combined, a five- or six-member heteroalicyclic ring containing atleast one nitrogen; or a physiologically acceptable salt thereof.
 2. Thecompound or salt of claim 1 wherein R¹ is selected from the groupconsisting of alkyl, cycloalkyl, alkenyl and alkynyl.
 3. The compound orsalt of claim 2 wherein R³ is selected from the group consisting ofhydrogen, alkyl, cycloalkyl and aryl.
 4. The compound or salt of claim 3wherein R⁴ is hydrogen.
 5. The compound or salt of claim 2 wherein Z isoxygen.
 6. The compound or salt of claim 5 wherein R⁵, R⁶, R⁸ and R⁹ arehydrogen.
 7. The compound or salt of claim 6 wherein R⁷ is selected fromthe group consisting of trihalomethyl and trihalomethanesulfonyl.
 8. Thecompound or salt of claim 7 wherein R⁵, R⁸ and R⁹ are hydrogen.
 9. Thecompound or salt of claim 8 wherein R⁶ and R⁷ combine to form amethylenedioxy or a 1,3-dioxano group.
 10. A method for the treatment ofa disorder characterized by inappropriate protein tyrosine kinaseactivity comprising administering to an organism afflicted with such adisorder a therapeutically effective amount of one or more compounds orphysiologically acceptable salts of claim
 1. 11. The method of claim 10wherein said therapeutically effective amount of said compound or saltof claim 1 is administered as a pharmacological composition.
 12. Apharmacological composition comprising: a compound or salt of claim 1;and, a physiologically acceptable carrier or exipient.
 13. The method ofclaim 10 wherein said organism comprises a mammal.
 14. The method ofclaim 10 wherein said organism is a human.
 15. The method of claim 10wherein said disorder is selected from the group consisting of braincancer, colon cancer, prostate cancer, kidney cancer, breast cancer,lung cancer, salivary gland cancer, oral cancer, pancreatic cancer,bladder cancer, Kaposi's sarcoma, melanoma and ovarian cancer.
 16. Themethod of claim 10 wherein said disorder comprises a skeletal disorder.17. The method of claim 10 wherein said disorder comprises a fibroticdisorder.
 18. The method of claim 10 wherein said disorder comprises ablood vessel proliferative disorder.
 19. The method of claim 17 whereinsaid fibrotic disorder is selected from the group consisting ofrestinosis, hepatic cirrhosis, glomerular sclerosis, interstitialnephritis, interstitial pulmonary fibrosis, atherosclerosis, woundscarring and scleroderma.
 20. A method of inhibiting the metastasis of acancer comprising administering to an organism in need of suchinhibition a therapeutically effective amount of one or more compoundsor salts of claim
 1. 21. The method of claim 20 wherein saidtherapeutically effective amount of one or more compounds or salts ofclaim 1 is administered as a pharmacological composition.
 22. The methodof claim 20 wherein said cancer is selected from the group consisting ofcolon cancer, prostate cancer, pancreatic cancer, Kaposi's sarcoma,ovarian cancer, breast cancer and gliomas.